Anti-deoxyribonucleic acid (DNA) antibodies in the clinical laboratory are intimately linked to the diagnosis and monitoring of systemic lupus erythematosus (SLE); however, the characteristics of the analytical methods and the properties of the antibodies themselves are heterogeneous. To review the definition and properties of anti-double-stranded anti-DNA (anti-dsDNA) antibodies, the adequacy of analytical methods, and the clinical requirements for this biomarker. Through PubMed we searched the existing literature with the terms anti-dsDNA, editorial, review, guideline, meta-analysis and SLE. The last search, anti-dsDNA and SLE restricted to the last two years. Information was expanded through related articles and those published in official state bodies related to anti-dsDNA and SLE. Clinical laboratory methods for anti-dsDNA analysis and their characteristics are analyze. The clinical utility of anti-dsDNA in its diagnostic, clinical association and follow-up aspects of SLE is reviewed. There is wide variability in analytical methods and deficits in standardization persist. They are part of the current SLE classification criteria and are used as markers in the follow-up of the disease. Their diagnostic usefulness improves when they are determined in antinuclear antibody (ANA)-positive patients. In follow-up, quantification is of interest, preferably with the same analytical method (given the deficits in standardization). Systemic autoimmune rheumatic diseases have a low prevalence and are associated with a variety of signs and symptoms that make diagnosis challenging. Effective diagnostic biomarkers such as antibodies against anti-deoxyribonucleic acid have not yet been identified (DNA) .Anti-dsDNA (anti-double-stranded DNA) antibodies have the ability to recognize all DNA structures present in chromatin, either relaxed or active. Thus, these antibodies recognize DNA, linear single-stranded DNA, double-stranded DNA (dsDNA) either circular or helical in its different forms. The most frequent forms, B dsDNA, with a right-handed double helix; Z dsDNA, with left-handed rotation; dsDNA, which is elongated with a long helix; and supercoiled helical dsDNA . HoweverThere is a wide spectrum of laboratory techniques available for the determination of anti-dsDNA, which challenges the use and interpretation of anti-dsDNA test results. The objective of this study is to review the definition and properties of anti-dsDNA antibodies, analyze the analytical and clinical properties of the measurement techniques available, and develop tools that facilitate the use of anti-dsDNA as a biomarker in systemic lupus erythematosus (SLE) patients.A literature search of articles was performed on PubMed using the term anti-dsDNA and by type of publication, considering editorials, reviews, guidelines, or meta-analyses. A search of articles on anti-dsDNA and SLE restricted to the last two years was also carried out. A supplementary search for related articles and material on anti-dsDNA and SLE released by national entities was finally performed.The information gathered is structured into five sections, namely: definition of anti-DNA antibodies and anti-dsDNA, properties, determination, clinical utility, and guidelines for their use.DNA is the main component of genetic material and stores genetic information. In eukaryotes and prokaryotes, DNA is compacted into a structure called nucleosome, which is composed of DNA and histones .Anti-DNA antibodies recognize all DNA structures or components. However, in clinical terms and despite its limitations, the most relevant autoantibodies are anti-dsDNA .SLE patients generate anti-dsDNA antibodies either when isolated or linked to proteins  or integrated into more complex structures such as nucleosomes .Antibodies against nucleosomes and histones have clinical relevance. Whereas anti-histone antibodies are associated with drug-induced lupus, the clinical role of anti-nucleosome antibodies is similar to that of anti-dsDNA antibodies and can be detected in the early stages of SLE, with a notably lower clinical implantation , 7.When a humoral response is initiated, the affinity of antibodies is low; however, as it progresses, the affinity of the antibody to its ligand (epitope) increases. In multi-functional molecules such as immunoglobulins, the strength with which immunoglobulin interacts with the antigenic molecule progressively increases via different ligands (avidity) . The matIn a normal immunogenic context, a bacterial or viral infection can induce a transient immune response that recognizes the anti-dsDNA antibodies against the bacterium or virus and even of the host itself . If the Concurrently to the infectious agent, other stimuli  can generate anti-dsDNA antibodies in patients with SLE, cancer, or other autoimmune diseases , 11], , . There iIn the past, anti-dsDNA antibodies were detected by enzyme-linked immunosorbent assay (ELISA) , CrithidThe most widely spread techniques in our environment are FEIA and CLIFT, followed by EIA, chemiluminescent immunoassay (CLIA), and MPIA, according to the methodology assessment reports of the quality program UK-NEQAS, with more than 600 participants . The useFarr’s technique, described in 1969  is consiIn CLIFT, native dsDNA is strongly compacted in the kinetoplast. It has a high specificity and a high positive predictive value. However, its sensitivity is lower than that of other methods  .In the ’80s, completely automated methods started to spread, first ELISA, then FEIA, MPIA, and CLIA. These methods have a higher sensitivity and lower specificity than CLIFT (it detects anti-dsDNA antibodies of lower affinity and avidity), with varying characteristics across manufacturers. High-salt concentrations such as wash buffer are used in ELISA  and CLIAIn the last years, a new flow cytometry-based method has been developed for detecting free anti-DNA autoantibodies and anti-DNA integrated into circulating immune complexes (endogenous DNA-anti dsDNA), with the latter having been integrated into routine screening and determination methods .The characteristics of the most widely spread methods are shown in With respect to the substrate, CLIA , 31 and The conjugate generally allows for the detection of IgG antibodies. However, there are some exceptions, as some EIA analyzers detect IgG and IgM anti-dsDNA antibodies  , whereas–The analysis takes longer with EIA and FEIA, as compared to MPIA and CLIA. When CLIFT is performed manually, the duration of the process is 60 min, although this time varies with automation and the time needed to read the sample by fluorescence microscopy.–FEIA, MPIA, and CLIA methods enable continuous sample loading; whereas EIA and CLIFT work in lots.–Calibration curves are stable during a variable period of time in FEIA, MPIA, and CLIA, whereas EIA requires a curve per lot, and no calibration curve is generated by CLIFT (a negative and positive control is performed for each lot).–Detection is quantitative in all methods except for CLIFT, which is semi-quantitative with serial sample dilutions. Nevertheless, results can be expressed qualitatively by any of the methods used (positive/negative) by using a cut-off point established by each laboratory or manufacturer.–The analytical range varies across manufacturers, with it being wider in CLIA methods.Cut-offs and reference values are established by each laboratory. The table shows reference values provided by manufacturers. Although all methods are based on an international standard, there is persistent variability in cut-offs and reference intervals, which demonstrates a lack of standardization.Comparative study of the analyzers used in the different methods analyzed demonstrates a moderate-to-high level of agreement among them, with kappa coefficients ranging from 0.47 and 0.68; clinical sensitivities for SLE between 5.7 (CLIFT) and 33.3% (EIA); specificities between 89.8 (MPIA) and 98.8% (CLIFT); positive likelihood ratio between 2.93 (MPIA) and 17.6 (CLIFT); and a non-significant negative likelihood ratio for any of the methods ranging between 0.71 (EIA) and 0.96 ..35].Therefore, for the diagnosis of SLE, the Spanish Ministry of Health and Consumer Affairs recommends that anti-dsDNA antibodies are measured by CLIFT in a 1:10 dilution in patients with antinuclear antibodies . CLIFT cIn addition, guidelines recommend that the method and reference values used for healthy controls and SLE patients are detailed in the laboratory report , 37.In the presence of symptoms of SLE, a positive antinuclear antibody test, and an elevated anti-dsDNA titer, SLE is the first diagnostic option . Once thIn active lupus nephritis, a recent study comparing Farr, ELISA, FEIA, and CLIA demonstrate a level of agreement of 95% between Farr and FEIA. With respect to lupus activity, Farr and FEIA showed a similar behavior .The first international standard for anti-dsDNA, Wo/80 was published by the World Health Organization (WHO) in 1985 . This rehttps://www.nibsc.org/products/brm_product_catalogue/detail_page.aspx?catid=15/174) [The standardization panel and the WHO have prepared and validated a new reference reagent, the so-called anti-dsDNA reference reagent for lupus (oligo-specific) 15/174. It has a nominal power of 100 units/ampoule, but it is not equivalent to the first international standard (Wo/80) and cannot be considered a continuation. It is available at NIBSC (=15/174) .As shown in In SLICC 2012 criteria, anti-dsDNA antibodies were analyzed by immunoassay, CLIFT, and ELISA. The anti-dsDNA criterion was associated with an SLE with a sensitivity of 57.1% and a specificity of 95.9% in a sample of 716 patients with different autoimmune or inflammatory diseases . In juveIn the 2019 ACR–EULAR criteria , 22 critNumerous studies have demonstrated that anti-dsDNA are pathogenic in lupus nephritis (LN) and are strongly associated with the disease . Anti-dsThe clinical association with LN increases when anti-dsDNA antibodies co-exist with anti-nucleosomes and anti-histones, which contributes to differentiate SLE with LN from SLE without LN, which increases LN severity . Other aSome guidelines recommend LN response to treatment to be monitored by anti-dsDNA and complement test, urine sediment test, proteinuria in 24 h urine or protein/creatinine ratio, and serum creatinine . HoweverAnti-dsDNA and C3–C4 complement concentrations (C3/C4) are serological markers of SLE activity. Thus, to assess SLE activity, it is recommended that anti-dsDNA titers and levels of C3/C4 are both assessed . NeverthPregnancy is associated with alterations in C3/C4 concentrations. However, the determination of C3/C4 and anti-dsDNA concentrations during pregnancy is useful for monitoring lupus activity. This test must be performed upon suspicion of lupus activity . AndreolBritish Isles Lupus Assessment Group Index) and BILAG 2004, ECLAM (European Consensus Lupus Activity Measurement), SLAM (Systemic Lupus Activity Index), SLAQ (Systemic Lupus Activity Questionnaire) that not always include anti-dsDNA elevation and hypocomplementemia among its descriptors [Anti-dsDNA antibodies and C3/C4 are included in SLE activity indices. criptors . In 2016criptors .At present, efforts are focused on establishing a definition to differentiate reduced SLE activity from remission. Complete remission is established when SLE is inactive without medication or only with anti-malaria drugs such as hydroxychloroquine. For the moment, a definition of reduced activity has not yet been established, and some definitions consider SLEDAI-2K and the drugs needed to maintain reduced SLE activity. Definitions have been provided for minimal SLE activity (MDA), low SLE activity (LDA), and low SLE activity status (LLDAS). Serological activity (measured based on anti-dsDNA and C3/C4) is included in LLDAS. In contrast, the definition of MDA and LDA only considers clinical variables .Recently, the Mexican Association of Rheumatology recommends that low complement levels, elevated levels of anti-dsDNA, and slightly increased C-reactive protein are considered to determine whether the presence of fever is associated or not with SLE activity in a patient .A variety of indices are available to assess response to treatment . HoweverIn this last section, the authors provide guidelines for an adequate use of anti-dsDNA based on available evidence.The clinical and pathogenic interest of anti-dsDNA increases when anti-dsDNA of high affinity and avidity are detected. In general, high-avidity and high-affinity antibodies are detected with Farr (unused) and CLIFT, which have a high specificity. Therefore, the use of CLIFT is recommended for the diagnosis of SLE. Nevertheless, this technique can be used as a second-line technique after a positive quantitative test. If CLIFT cannot be used, a test result exceeding two times the upper limit of the reference value for the control group will be considered positive. The clinician should be familiarized with the technique and laboratory reference values.For the diagnosis and upon clinical suspicion of SLE, anti-dsDNA should be determined in patients with positive antinuclear antibodies, and results for the two types of antibodies should be reported. Exceptionally, on high suspicion of SLE, anti-dsDNA can be directly measured because anti-dsDNA can be detected in patients negative for antinuclear antibodies based on the technique and cut-off used, as fixation methods and the characteristics of analytical methods may vary. For example, in HEp-2 cells, dsDNA is found in nuclei bound to histones and other nuclear proteins and fixed to the glass plate using organic solvents. In contrast, in specific methods, native purified or recombining anti-dsDNA is bound to a solid plastic plate without using any substance.Anti-dsDNA antibodies only have diagnostic value in SLE and test results should always be interpreted in relation to other clinical data. Anti-dsDNA antibodies have high specificity and are included in immunological classification criteria. An elevated concentration of anti-dsDNA in people with clinical signs of SLE and a positive antibody test is suggestive of SLE as the first diagnostic option. However, a negative result does not exclude SLE, as it has a low sensitivity and negative likelihood ratio.Quantitative anti-dsDNA determination is recommended – always using the same method – for monitoring SLE patients.Anti-dsDNA is associated with lupus activity, especially when detected at high concentrations using high-specificity methods and are concurrent to other clinical or analytical signs of SLE activity. Elevated levels of anti-dsDNA alone are not predictive of SLE activity.Anti-dsDNA and complement C3/C4 levels are included in SLE activity indices and should be both determined on suspicion of SLE activity, also in pregnant SLE patients.Anti-dsDNA is correlated with LN and has a relative utility for diagnosis and assessment of SLE activity. As it occurs with SLE activity, high concentrations and specificity improve the association and clinical utility of anti-dsDNA in LN. However, test results should be interpreted in relation to other kidney disease markers.There is a wide range of analytical methods for the determination of anti-dsDNA antibodies but a lack of standardization. Not all methods detect antibodies with the same level of avidity and affinity. Anti-dsDNA with high avidity and affinity in antinuclear antibody (ANA)-positive patients have a high diagnostic specificity for SLE. Anti-dsDNA determination – always using the same technique – is useful for follow-up of SLE (because of the lack of standardization). Finally, variability and progress in the measurement methods used modify their clinical characteristics.


Post-stroke delirium (PSD) after intracerebral hemorrhage (ICH) is considered to be even more detrimental compared to that after ischemic stroke. Treatment options for post-ICH PSD remain limited. This study aimed at investigating to what extent prophylactic melatonin administration may have beneficial effects on post-ICH PSD. We performed a mono-centric, non-randomized, non-blinded, prospective cohort study, including 339 consecutive ICH patients admitted to the Stroke Unit (SU) from December 2015 to December 2020. The cohort consisted of ICH patients who underwent standard care (defined as the control group) and ICH patients who additionally received prophylactic melatonin  within 24 h of ICH onset until the discharge from the SU. The primary endpoint was post-ICH PSD prevalence. The secondary endpoints were: (i) PSD duration and (ii) the duration of SU stay. The PSD prevalence was higher in the melatonin treated cohort compared to the propensity score-matched (PSM) control group. Post-ICH PSD patients receiving melatonin had shorter SU-stay durations, and shorter PSD durations, although not statistically significant. This study shows no efficacy in limiting post-ICH PSD with preventive melatonin administration. Post-stroke delirium (PSD) represents a severe complication of intracerebral hemorrhage (ICH) ,2,3. TheThe pathogenetic processes behind the development of post-ICH PSD are poorly understood. Defective neuronal connectivity and neurotransmission, impaired brain metabolism, neuroinflammation, disturbed sleep–wake cycles, abnormal oxygen supply, electrolytic disturbances, overt stress responses, altered cellular signaling, and brain vascular dysfunction are considered to be implicated in PSD development ,13,14. DMelatonin is a hormone secreted by the pineal gland, and in clinical practice exogenous melatonin supplementation is used mainly for, but not limited to, treating sleep disorders . The hypWith respect to the therapeutic efficacy of melatonin, conflicting data have been reported. Previous studies have indicated that melatonin supplementation may be effective in preventing delirium, however, there are also studies reporting no benefit thereof ,23. ExogIn view of the former considerations, we hypothesized that preventive administration of melatonin will decrease the frequency of post-ICH PSD and also demonstrate beneficial effects on related outcomes . As such, the aim of the present study in a cohort of ICH patients was to examine the effect of preventive administration of melatonin on post-ICH PSD prevalence. We also examined the effect of preventive administration of melatonin on PSD duration and SU-stay duration.We conducted a mono-centric, non-randomized, non-blinded, prospective cohort study. Our cohort consisted of ICH patients who were prophylactically treated with melatonin for PSD prevention versus non-treated ICH patients (defined as the control group). The institutional ethics committee approved the protocol of the study (protocol number 752/2018BO2). Due to the clinic-wide consent regarding the use of de-identified routine treatment data for research purposes, the individual informed consent from the participants was waived.Consecutive ICH patients admitted to the stroke unit (SU) of the University Hospital of Tübingen, between 1 December 2015 and 31 December 2020 were included. All patients were diagnosed with nontraumatic ICH according to the International Classification of Diseases, 10th Revision I61.The following exclusion criteria were applied: (1) duration of SU stay <24 h; (2) diagnosis of delirium caused by benzodiazepines on admission (oa); (3) a Richmond Agitation-Sedation Scale (RASS) level of −5 or −4 for the majority (>50%) of the stay; (4) patients on mechanical ventilation or in shock or who had severe liver or renal insufficiency; (5) an incomplete record of the RASS and Intensive Care Delirium Screening Checklist (ICDSC) during the stay; (6) patients who underwent neurosurgical intervention immediately after baseline-CT; (7) patients with traumatic ICH.All treated patients underwent prophylactic melatonin supplementation according to the standard operating procedure (SOP) of the SU for PSD prevention, which has previously been described in detail .In brief, the rationale for preventive application of melatonin in the SOP were: (i) the lack of clear evidence regarding therapeutic options for PSD prevention and management; (ii) melatonin’s excellent tolerability and safety profile; and (iii) evidence regarding the beneficial effect of melatonin in delirium prevention in elderly, in stroke patients, and in ICU patients ,25,26,27The baseline characteristics, personal medical history, and in-hospital clinical parameters for all the participants were retrieved from the clinical information system . Data from head neuroimaging (CT or MRI) on admission were also obtained. We applied the ICDSC in order to assess PSD in all participants (both melatonin-treated and control cohort) . The ICD3), invasive procedures , history of diabetes mellitus (DM), history of hypercholesterolemia (HCL), history of atrial fibrillation (AF), history of coronary artery disease (CAD), obesity (defined as body mass index [BMI] > 30), chronic renal failure, chronic hepatic failure, history of smoking, history of alcohol consumption, history of malignancy, history of depression, cognitive status ), infections during stay at SU, NIHSS oa, presence of aphasia oa, ICH-score oa, modified Rankin scale (mRS) before the index event, the etiology of ICH , mass (benign and non-benign), oral anticoagulant (OAC), vessel pathology, other/unknown), hemorrhagic location at baseline (deep white matter (DWM), lobar, brainstem, cerebellum, left or right hemisphere), intraventricular hemorrhage (IVH) oa, IVH extension, ICH volume  with a match tolerance of 0.2 was performed in order to balance baseline differences in clinical covariates between patients treated with melatonin and controls. Standardized mean differences (SMD) were calculated to compare participant features after PSM. SMD values greater than 0.25 were considered indicative for imbalance ,32.We used the Shapiro–Wilk test to check the distribution of the data. For categorical variables, the differences between groups regarding demographics and clinical characteristics were assessed using Pearson’s chi-squared test. For continuous variables, we applied Mann–Whitney U tests due to non-normal distribution of the data. Values are expressed as total number (n) with the respective percentage (%) for categorical variables. For continuous variables, values are expressed as medians with the respective interquartile ranges (IQR).We used chi-square tests to examine the difference in post-ICH PSD prevalence, between the melatonin-treated cohort and the control group. To better capture the treatment effect on each group (for descriptive purposes) between group differences regarding PSD duration and SU-stay duration, we applied Mann–Whitney U tests. For the effect of the preventive administration of melatonin on the PSD duration (hours) and SU-stay duration (hours), we applied linear regression analyses, using melatonin treatment as predictor, after logarithmic transformation of the dependent variables  due to non-normal distribution of the data. Adjusted models for the variables that differed between groups or correlated with the outcome of interest in univariate analysis, were also performed. The multicollinearity was assessed by using the variance inflation factor (VIF). VIF values of 10 were considered as cut points for the elimination variable from the model . No multFor the statistically significant results, we also performed sensitivity analyses by excluding patients that died during hospitalization, aiming to eliminate any possible latent effect of this factor on the PSD duration or SU-stay duration. p < 0.05) was set as the level of statistical significance for all procedures. All statistical analyses were performed with SPSS .An alpha error of 5%  developed PSD during the SU stay. The majority of them (74.2%) developed PSD within the first 48 h of admission, whereas the vast majority of them (∼90%) developed PSD within the first 120 h after admission . Median The melatonin-treated cohort had lower NIHSS, ICH-scores, and ICH-volume oa, increased prevalence of HCL, OAC as etiology of ICH, and decreased prevalence of EVD, surgical evacuation, and IVH extension compared to the control group. After the PSM, no statistically significant between-group differences in characteristics were noted, with the exception of brainstem location of ICH at baseline, which was higher in the control group. Patients’ characteristics, based on receiving (or not) melatonin, are presented in p = 0.004), and compared to the PSM control group .The PSD prevalence was higher in the melatonin-treated cohort (47.9%) compared to the control group  melatonin after the PSM in the initial sample (n = 88) are presented in p = 0.088) nor adjusted models . Adjustment was made for variables that significantly correlated with PSD duration, as presented at p-value for Mann–Whitney U test = 0.104). Box plots presenting data for PSD duration (hours) with respect to administration (or not) of melatonin are shown in In PSD patients, preventive melatonin administration did not correlate with duration of PSD in neither crude . The significance did not remain in the adjusted model . Adjustment was made for variables that significantly correlated with SU-stay duration, as presented in p-value for Mann–Whitney U test = 0.033). Box plots presenting data for SU-stay duration (hours) in respect to administration (or not) of melatonin in PSD are shown in Melatonin administration was negatively correlated with SU-stay duration of PSD patients , although post-ICH PSD patients receiving melatonin had lower SU-stay duration compared to the control group (187 h vs. 274 h)  . p = 0.941) and adjusted  models. Adjustment was made for variables that significantly correlated with SU-stay duration, as presented in p-value for Mann–Whitney U test = 0.910). Box plots presenting data for SU-stay duration (hours) in respect to administration (or not) of melatonin in non-PSD are shown in Post-ICH without PSD patients’ characteristics, based on receiving (or not) melatonin after the PSM in the initial sample (n = 149), are presented in Results regarding the effect of preventive administration of melatonin on SU-stay duration  are summarized in In this cohort study, we investigated the potential preventive effect of low-dose melatonin on PSD. The PSD prevalence was higher in the melatonin cohort compared to control group. Since age of treated patients was slightly higher compared to controls after PSM (76 vs. 74.5 years), it may comprise a possible reason for these results. Moreover, the lack of a preventive effect of melatonin on PSD prevalence may be attributed to the low melatonin dosage in our study, especially when it is compared to melatonin dosages of previous studies reporting a beneficial effect of melatonin on ICH ,34.The preventive administration of melatonin had marginal beneficial effects on SU-stay duration and on PSD duration in post-ICH patients. More precisely, the post-ICH patients receiving melatonin appeared to have shorter, but not statistically significant, PSD duration compared to untreated patients. Of note, in our study, median SU-stay duration of PSD patients receiving melatonin was shorter compared to the PSD control group, highlighting a possible beneficial effect of the preventive melatonin administration in ICH patients. In particular, the median SU-stay duration of PSD patients receiving melatonin was reduced by more than 3.5 days compared to their non-treated PSD counterparts. Considering this, we could hypothesize that melatonin may have a beneficial effect only on specific PSD parameters, while higher melatonin dosages may be required for preventive effects on PSD prevalence to become evident. The vast majority of post-ICH PSD patients (~90%) developed PSD within the first 120 h after admission. By contrast, one previous report  suggestsAmong the strengths of our study is the inclusion of a large number of well-characterized ICH patients, following a standardized protocol for PSD detection. Secondly, we performed multivariate analyses with adjustment for a large amount of potential cofounding factors, sensitivity analyses for the statistically significant results, and mainly a PSM analysis in an attempt to minimize any possible selection bias. At this point we must also acknowledge the limitations of our study. Firstly, we did not randomize the sample for melatonin administration and the generalizability of our results is limited, despite our approach of using PSM analysis. Thus, the present findings require validation from a prospective randomized controlled trial (RCT). Moreover, given the different pathophysiological mechanisms implicated in different PSD subtypes , additioThis study shows no efficacy in limiting post-ICH PSD with preventive melatonin administration, as the PSD prevalence was higher in the melatonin cohort. However, the preventive administration of melatonin appears to have marginal beneficial effects on SU-stay duration and on PSD duration in post-ICH PSD. Yet, due to methodological limitations related to the non-randomized study design, we cannot totally exclude that the utility of melatonin may have been underestimated. Taking into consideration the limited available therapeutic strategies for PSD, large-scale RCTs are warranted to further test and validate the role of melatonin in preventing and treating post-ICH PSD.


Mycobacterium avium subsp. paratuberculosis (MAP) causes bovine paratuberculosis (PTB). PTB is responsible for significant economic losses in dairy herds around the word. PTB control programs that rely on testing and culling of test-positive cows have been developed. Current diagnostics, such as ELISA for detecting MAP antibodies in serum samples and PCR detecting MAP DNA in feces, have inadequate sensitivity for detecting subclinical animals. Innovative “omics” technologies such as next-generation sequencing (NGS) technology-based RNA-sequencing (RNA-Seq), proteomics and metabolomics can be used to find host biomarkers. The discovered biomarkers  can then be used to develop new and more sensitive approaches for PTB diagnosis. Traditional approaches for measuring host antibodies and biomarkers, such as ELISAs, northern blotting, quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR), cDNA microarrays, and mass spectrometry are time-consuming, expensive, and sometimes exhibit poor sensitivity. With the rapid development of nanotechnology, low-cost monitoring devices for measuring antibodies against MAP proteins in point-of-care (POC) settings have been developed. Lateral flow assays (LFAs), in particular, are thought to be appropriate for the on-site detection of antibodies to MAP antigens and/or host biomarkers. This review aims to summarize LFAs that have recently been developed to accurately detect antibodies against MAP antigens, as well as the benefits that host biomarkers linked with MAP infection give to PTB diagnosis. The identification of these novel biomarkers could be the basis for the development of new LFAs. The dairy industry and producers are likely to benefit from reliable and rapid technologies capable of detecting MAP infection in situ to establish a quick and sensitive PTB diagnosis. In cattle, MAP infection induces a chronic wasting disease characterized by diarrhea and progressive loss of body condition  and negative controls (N = 138)  technology-based RNA-sequencing (RNA-Seq), proteomics and metabolomics have immense potential since they enable the study of PTB pathogenesis and can be used to discover biomarkers for the development of innovative diagnostic tools, drugs, or vaccines . TranscrN = 138) . ImportaN = 27), subclinical non-shedder (N = 50), and clinical shedders (N = 18) than in a healthy control group (N = 11) from a PTB-free farm. Even though the study included a reduced number of healthy control cows due to the difficulty in finding a PTB-free farm, A2M ELISA demonstrated superior diagnostic performance (90.4% sensitivity and 100% specificity) than two commercial ELISAs for the detection of anti-MAP antibodies.The differences in host protein concentrations/levels in blood and feces in response to MAP infection may be of diagnostic value. Various proteomic platforms have been explored. Using iTRAQ, a liquid chromatography and tandem mass spectrometry (LC–MS) approach, Seth et al. found transthyretin, retinol-binding proteins, and cathelicidin in serum samples from MAP-infected animals . You et Biomarkers include host non-coding RNAs (ncRNAs) as well as mRNAs, proteins or metabolites. MicroRNAs are one of the most studied types of ncRNAs. MicroRNAs (18–25 nucleotides long) are highly conserved small RNAs that primarily regulate gene expression by lowering the stability of their target mRNAs . BecauseN = 356) were followed up annually for 2–4 years using direct analysis in real time coupled with high resolution mass spectrometry (DART-HRMS)  . InfecteRT-HRMS)  exhibiteRT-HRMS) . discoveRT-HRMS) . Sera weRT-HRMS)  were sho4.Current PTB diagnostics require transporting the biological sample from the farm to the laboratory, as well as highly trained laboratory personnel and multistep preparation. As a result, the interval between sampling and outcome is long. Because of the rapid development of nanotechnology, it is now possible to develop quick and inexpensive monitoring devices that can aid in the measurement of host antibodies, microRNAs, and proteins in POC settings or resource-limited facilities , 50. TheNanoparticles (NPs) offer unique qualities such as high surface area to volume ratio, high surface energy, tunable absorption and emission properties, high stability, and biocompatibility, making them ideal for the designing of POC platforms . BecauseBecause of their innate biocompatibility, low cytotoxicity, and high stability in biological fluids, AuNPs are excellent materials for detecting host antibodies, microRNAs, and proteins in biological samples. AuNPs can be easily functionalized with biomolecules that have high affinity for these analytes. Adsorption-based, covalently biding-based, and affinity-based approaches are the most often used methods for functionalizing the surface of AuNPs. The adsorption-based method is based on electrostatic or hydrophobic interactions between the ligand and the surface of the AuNPs. The ligand is adsorbed onto the surface of the AuNPs, forming a non-covalent bond. In the covalent binding-based method, the ligand is covalently linked onto the surface of the AuNP using a thiol group. This can be accomplished by directly conjugating a sulfur-containing molecule or using a bi-functional linker. The linker has a thiol group at one end that has a high affinity for the AuNP surface, resulting in a semi-covalent bond (S-Au), and another functional group at the other end that allows other biomolecules to be attached. The affinity-based surface modification method involves functionalizing the AuNP surface with moieties that provide specific binding sites for biomolecule coupling. These affinity sites allow the selective attachment of biomolecules to the AuNP surface.In the case of microRNAs, AuNPs are often functionalized with oligonucleotides containing a domain complementary to the microRNA’s target sequence. In the case of protein detection, AuNPs are functionalized with specific antibodies against the target protein. AuNP-based biosensors are categorized into two types: solution-based biosensors and solid support-based biosensors. The first describes biosensing devices that use AuNPs dispersed in solution . In the latter group, AuNPs are attached to a solid support .5.Lateral flow assays (LFAs) are diagnostic assays based on the principles of immunochromatographic lateral flow test strips. The test sample flows along a solid substrate through capillary action, which takes 10–15 min to complete. LFAs are quick assays, which only require a few drops of a sample diluted in a buffer. LFAs are made up of a membrane (frequently made of nitrocellulose), a sample pad (made of cellulose or glass fiber), a conjugate pad (made of glass fiber), and an absorbent or wicking pad , all of which are laminated into a sheet of plastic known as the backing card .When a test sample containing the target analyte (for instance a protein) is added to the sample pad, it flows through the membrane solubilizing the conjugate (an AuNP-antibody) that flows alongside it . The conIn the case of microRNA detection, NALFAs are typically sandwich assays that rely on the complementary binding between the target microRNA sequence in the sample and both membrane-immobilized and AuNP-labeled oligonucleotide probes. The probes are immobilized in the membrane by high-affinity interactions between protein pairs, such as biotin-avidin/streptavidin. Typically, two biotinylated DNA probes, the capture and the control probes, are conjugated with streptavidin and distributed on the nitrocellulose membrane, forming the test and control lines, respectively. A detection probe complementary to the 3′ half of the target microRNA is conjugated to AuNPs via its thiol group, and this DNA-AuNPs conjugate is loaded on the conjugate pad. When the microRNA-containing sample is applied to the sample pad, it travels through the membrane until it reaches the conjugate pad, where the target microRNA hybridizes with the AuNPs-conjugated capture probe. The sample continues to migrate and is captured by the control probe in the control line. The accumulation of target produces a red band in the test line, the intensity of which is proportional to the amount of target microRNA present in the sample. In the absence of the microRNA, just the control band is observed, ensuring proper liquid flow through the NALFA. MicroRNA detection for POC diagnosis has the disadvantage of requiring microRNA isolation from biological fluids before detection. Emerging technologies for microRNA extraction and detection in portable devices, on the other hand, should increase the current usage of microRNAs for diagnosis .6.Novel LFAs for the fast detection of MAP infection have been summarized in An AuNPs-based LFI for the detection of specific antibodies against MAP2963 protein was developed . In thisJain et al. developed a LFI based on a cocktail of MAP 2677c, 3547c, 4308c, 1693c, and 2168c recombinant proteins (each antigen at 2 mg/mL in the cocktail) . The MAP7.Although POC devices for human medicine have made tremendous progress, due primarily to the COVID-19 pandemic, animal POC testing for farm animals has yet to reach its full potential. LFAs could be used to monitor large numbers of animals in the field and to detect antibodies against MAP antigens, as well as bovine microRNAs, proteins or metabolites associated with MAP infection using a relatively small sample volume. Although LFAs for detecting antibodies against MAP antigens have been developed, the next generation of host biomarkers-based LFAs with greater sensitivity for detecting subclinical cases is expected to be developed. Furthermore, multiplex LFAs would enable the simultaneous detection of more than one discriminative biomarker, increasing the detection of the different stages of the infection. In the future, it is expected that mass production techniques would significantly reduce the cost of manufacturing LFAs, making this technology affordable to producers and veterinarians. Besides PTB, LFAs would be especially useful for diagnosing zoonotic mycobacterial infections like bovine tuberculosis which is endemic in low- and middle-income countries with limited access to sophisticated laboratories. Moreover, the development of host biomarker signature based LFAs that are ideally DIVA  could be a promising path ahead. The future application of LFAs will require previous evaluation and validation on larger animal cohorts of experimentally and naturally infected cows.M-AH: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing. AB: Formal analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – review & editing. AN: Investigation, Methodology, Writing – review & editing. GB-B: Methodology, Software, Visualization, Writing – review & editing. RC: Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.


Many clinical trials have demonstrated the value of drug-coated balloons (DCB) for in-stent restenosis. However, their role in de novo lesions is not well documented. The aim of this study is to evaluate the safety and efficacy of the DCB-only strategy compared to other percutaneous coronary intervention strategies for de novo coronary lesions.  The PubMed, Embase, Web of Science, and Cochrane Library Central Register of Controlled Trials (CENTRAL) electronic databases were searched for randomized controlled trials published up to May 6, 2023. The primary outcomes were major adverse cardiac events and late lumen loss. P = 0.631) and a significant decrease in late lumen loss (standardized mean difference (SMD) = −0.29, 95% CI: −0.53 to −0.04, P = 0.021). This effect was consistent in subgroup analysis regardless of indication, follow-up time, drug-eluting stent type, and dual antiplatelet therapy duration. However, DCBs were inferior to DESs for minimum lumen diameter and percentage diameter stenosis. The DCB-only strategy showed significantly better outcomes for most endpoints compared to plain-old balloon angioplasty or bare metal stents.  A total of eighteen trials with 3336 participants were included. Compared with drug-eluting stents, the DCB-only strategy was associated with a similar risk of major adverse cardiac events (risk ratio (RR) = 0.90; 95% confidence interval (CI): 0.59 to 1.37,  Interventions with a DCB-only strategy are comparable to those of drug-eluting stents and superior to plain-old balloon angioplasty or bare metal stents for the treatment of selected de novo coronary lesions. Additional evidence is still warranted to confirm the value of DCB before widespread clinical utilization can be recommended. The devices and techniques for percutaneous coronary intervention (PCI) continue to evolve and have already revolutionized the treatment of coronary artery disease. The advent of plain-old balloon angioplasty (POBA) and bare metal stents (BMS) makes it possible for the invasive expansion of stenosed coronary vessels. However, these techniques are limited by complications, such as elastic recoil, abrupt vessel closure, or postprocedure restenosis . Drug-elDCBs were first introduced as a treatment for stent-related restenosis and were shown to have promising results. Based on sufficient evidence, DCBs have been recommended as the first-line treatment option for ISR by the European Guidelines . With thAlthough some previous meta-analyses studying the effect of DCBs with default BMS implantation did not seem to produce favorable results, meta-analyses addressing the efficacy and safety of DCB alone (with bailout stenting only) approach were relatively few. Therefore, the present meta-analysis is aimed at summarizing the available evidence and comparing the DCB-only approach with other PCI strategies for the treatment of de novo coronary lesions.CRD42020158856).The PubMed, Embase, Web of Science, and Cochrane Library Central Register of Controlled Trials (CENTRAL) electronic databases were searched from inception until May 6, 2023. All published randomized controlled trials (RCTs) that compared the DCB-only approach with other PCI strategies for the treatment of patients with de novo coronary artery disease were identified. A search algorithm was used with a combination of relevant terms. No filters or language restrictions were applied. The detailed search strategy for each database has been provided in Supplementary Table All eligible studies meeting the following inclusion criteria were selected: (1) randomized controlled trials, (2) comparing DCB-only approach  with a control treatment , (3) patients in the study had de novo coronary artery disease, and (4) availability of angiographic or clinical outcome data without follow-up duration restriction. For studies with more than one follow-up period, the longest available angiographic and clinical follow-up results were considered for analysis. Studies that employed routine stents in the DCB group, studies that are not yet finished, and those with incomplete baseline data or follow-up results were excluded.The primary endpoints were major adverse cardiac events (MACEs) and in-segment late lumen loss (LLL). Secondary endpoints included target lesion revascularization (TLR), all-cause death or cardiac death, myocardial infarction (MI), binary restenosis (BR), minimum lumen diameter (MLD), and percent diameter stenosis (DS%).The search process and data extraction were conducted by two independent investigators . Conflicts were discussed and resolved by consensus. A standardized database (Microsoft Excel) was used to extract details on study information , patient characteristics , PCI devices and strategies, angiographic measures at baseline, and outcomes of interest at follow-up. Qualities of the included studies were assessed by the Cochrane risk of bias assessment tool for RCTs .P values were < 0.05 in two-tailed tests. The heterogeneity among trials was assessed using Cochran's Q test and means of the I2 statistic [P values of < 0.10 or I2 > 50% were considered significant for heterogeneity. We used a mixed-effects model to synthesize data. Both the random-effects model (DerSimonian and Laird) and the fixed-effects model (Mantel-Haenszel) were used to perform the analyses. Considering the differences in the designs, populations, types of interventions, and treatment effects across studies, a random-effects model was given preference for this meta-analysis. Pooled risk estimates were also examined by a fixed-effects model to avoid small studies being overly weighted. Furthermore, it has been recommended that a predictive interval, which reflects the variability of the treatment effects over different settings, should be routinely presented in the random-effects meta-analysis [Statistical analysis was performed using the Stata version 12.0 software . Combined risk ratios (RR) with a 95% confidence interval (CI) and mean differences (MDs) with standard deviations were presented as summary statistics, and results were presented as forest plots. Results were considered statistically significant when tatistic . P valueanalysis . We haveP < 0.05 suggested positive evidence of bias. Sensitivity analysis was performed by excluding one trial at a time to assess the contribution of each individual study to the summary statistics. Subgroup analyses between the DCB and DES groups were conducted according to vessel diameter, follow-up duration, clinical diagnosis, DES type, and DAPT period.Publication bias was evaluated using a funnel plot as well as Egger's test , and P <n = 10), five trials compared DCB to POBA [A total of 2473 articles were identified, of which 18 trials  satisfied the outlined inclusion criteria and were included in the meta-analysis –37. A fl to POBA , 26, 27, to POBA , 28, 36.P = 0.631), whereas a higher risk of MACEs was found in the BMS or POBA group   .P = 0.021) or uncoated device groups   .The random-effects model was used for the analysis, and similar results were obtained by the fixed-effects model (Supplementary Table P = 0.705), death or cardiac death , and MI . When comparing DCB-only against the BMS/POBA group, DCBs significantly reduced the risks of TLR , death or cardiac death , and MI   .P = 0.748). However, a statistically significant increase in MLD , as well as a significant reduction of DS% , was observed in the DES-treated patient group compared with the DCB group. Compared to the POBA or BMS group, DCBs presented a significant reduction in BR , a significant increase in MLD , and a significantly lower DS%   .We used the random-effects model to perform the above analyses. No significant differences were found between the analytical results of the two effect models  duration. In subgroup analysis, no statistically significant difference was detected in MACEs between the DCB and DES groups. However, the advantage of DCB in reducing LLL tended to decrease with the increasing of vessel diameter and follow-up duration .The risk of bias assessment in each individual study is presented in Supplementary Figure P = 0.417 for MACEs and P = 0.111 for LLL).Publication bias was assessed by funnel plots for the primary endpoints. The funnel plots were substantially symmetrical according to a visual inspection  at observed endpoints. Results suggested that no study significantly influenced the overall estimates  for specific de novo lesions or clinical scenarios , the DCB-only strategy represented a more effective and safer treatment compared to POBA or BMS. (2) There were no significant differences in the primary outcomes between the DCB-only and the DES group; however, DESs were associated with more favorable angiographic endpoints such as MLD and DS%. (3) In subgroup analyses, the DCB-only strategy performed comparably to DES regardless of vessel diameter, follow-up duration, clinical diagnosis, DES type, and DAPT duration, but the advantage of DCB may dwindle as the increase of vessel diameter and follow-up duration.Although DES has become the main therapy for coronary artery disease, POBA and BMS may also play a part in specific anatomical or clinical settings. The PEPCAD-BIF trial is aimed at exploring the effect of the DCB-only strategy in distal main or side branches of bifurcation lesions. Results showed that compared to POBA, DCBs had a statistically significant reduction in LLL and lower risks of restenosis . The DEBNewer generation DES has been shown to be effective and has become the first choice for de novo coronary stenosis. However, some limitations, such as stent thrombosis, stent restenosis, or long-term DAPT, can have an adverse impact on the prognosis. Therefore, researchers are exploring DCB as a promising option for the treatment of de novo coronary artery disease. The DCB followed by routine BMS implantation (DCB+BMS) strategy has been widely investigated as a replacement for DES. However, the results have not been very compelling , 39. RecThe DCB-only strategy was first adopted in small coronary vessels. Although the earlier PICCOLETO trial failed to demonstrate the safety of DCB in small coronary vessels , the subIn addition, the DCB-only strategy also presented a potential advantage in other anatomical or clinical settings. The REVELATION trial showed noninferiority of DCB compared to the second-generation DES regarding clinical and angiographic endpoints in patients with STEMI . SimilarBy summarizing the available evidence, this study demonstrated that DCB showed comparable safety and efficacy with the DES treatment. Given the fact that DES is the mainstay of therapy for de novo lesions, we emphatically compared the safety and efficacy between DCB and DES in subgroup analysis. Although subgroup analyses of trials comparing DCB to DES have shown that the results were stable regardless of vessel diameter, follow-up duration, clinical diagnosis, DES type, and DAPT duration, it should be noted that the effects of DCB in reducing LLL were less evident in some subgroups, such as large coronary vessels and longer follow-up time. Additionally, the DES group appeared to be more favorable than the DCB-only group in terms of angiographic outcomes, showing a significant increase in MLD and a significant reduction in DS%. Accordingly, further randomized controlled trials are still required to demonstrate the long-term benefits of DCB in various types of de novo coronary lesions, especially in de novo lesions of large coronary vessels.Although there have been meta-analyses investigating the use of the DCB-only strategy in de novo lesions, most of them focused on specific indications, such as small vessels –48, largEven though the safety and efficacy of the DCB-only strategy in different settings of de novo CAD were substantiated by the present study, it is very important to perform careful and extensive lesion preparation before using DCB . ConventThe comprehensive analysis of different endpoints increased the robustness and credibility of the study conclusions. However, considering the lack of large studies, relatively short follow-up period, and insufficient reports of hard clinical endpoints, such as death and cardiac death, the safety of DCB should not be overestimated at this time. Moreover, some studies excluded patients receiving a bailout stent, making the result more favorable for the DCB group. Finally, heterogeneity may have resulted due to multiple types of comparators used in a single study.The DCB-only strategy was comparable to DES and superior to POBA or BMS in primary safety and efficacy endpoints for selected de novo coronary lesions. Further studies are warranted to fully elucidate the long-term benefits of DCB compared to DES in various de novo lesions before the more extensive use of DCB can be recommended.


Gastrointestinal cancers represent a major burden in oncology worldwide. As radiotherapy is a cornerstone of the treatment strategies, accurate treatment planning is necessary. Imaging modalities, such as positron emission tomography with computed tomography (PET/CT) and magnetic resonance (MRI) could improve target volume determination. This review summarizes the role of PET/CT and MRI in radiotherapy treatment planning for anal, esophageal, rectal and pancreatic cancer.Positron emission tomography with computed tomography (PET/CT) and magnetic resonance imaging (MRI) could improve accuracy in target volume determination for gastrointestinal cancers. A systematic search of the PubMed database was performed, focusing on studies published within the last 20 years. Articles were considered eligible for the review if they included patients with anal canal, esophageal, rectal or pancreatic cancer, as well as PET/CT or MRI for radiotherapy treatment planning, and if they reported interobserver variability or changes in treatment planning volume due to different imaging modalities or correlation between the imaging modality and histopathologic specimen. The search of the literature retrieved 1396 articles. We retrieved six articles from an additional search of the reference lists of related articles. Forty-one studies were included in the final review. PET/CT seems indispensable for target volume determination of pathological lymph nodes in esophageal and anal canal cancer. MRI seems appropriate for the delineation of primary tumors in the pelvis as rectal and anal canal cancer. Delineation of the target volumes for radiotherapy of pancreatic cancer remains challenging, and additional studies are needed. Gastrointestinal cancers, including colorectal, esophageal, gastric, pancreatic, hepatocellular and biliary tract cancers, are the most common cause of cancer mortality worldwide. The estimated number of new cases of gastrointestinal cancers in 2020 was 5,142,192, and colorectal carcinoma remains the third most common cancer worldwide, with an incidence of 1,931,518 cases in 2020. Pancreatic cancer is still a disease with poor prognosis with an estimated 466,003 death cases worldwide in 2020 [PET is a functional imaging technique involving the use of radioactive tracers—positron emitters attached to targeted biologically active molecules. To date, the most commonly used tracer in tumors of the gastrointestinal tract is 18-F-fluorodeoxyglucose (FDG), an analogue of glucose, which accumulates in tissues with a high metabolic turnover. Currently, FDG-PET/CT is considered an essential diagnostic method for the initial staging of esophageal and anal cancers, and it could also play a role in assessing the response to chemoradiotherapy ,6. ThereThe role of PET/CT in radiotherapy treatment planning is still under investigation; however, a better understanding of tumor biology with new functional imaging techniques could have an impact on the target delineation, a concept called biological target volume (BTV). BTV takes into account the metabolic, biochemical and physiological changes within a tumor, making it possible to deliver a higher dose of radiation to the BTV, rather than the whole tumor, in a process called dose painting. Delivering higher doses in radioresistant areas, such as a hypoxic region, could result in better tumor control ,9.Other PET tracers have been developed, including fluorine-18 fluoromisonidazole (F-MISO), which binds to the hypoxic areas of a tumor, and 3′-deoxy-3′-fluorothymidine (FLT), which is a measure of the tumor proliferation rate ,9. RecenOn the other hand, MRI utilizes the magnetic properties of hydrogen protons within a tissue to generate image contrast. It provides higher resolution and greater soft tissue contrast when compared to CT imaging for some tumor sites, for example pelvic tumors. However, MRI can be more challenging for other tumor sites, such as the upper abdomen or mediastinum, due to organ motion artefacts and the central location, which causes a reduced receiver coil sensitivity. Over the past decade, several technical innovations have improved the image artefacts, such as the use of automatic gating navigators or multi-channel receiver coils.Diffusion-weighted MRI (DWI) has already been widely adopted in cancer imaging due to its ability to enhance soft-tissue contrast ,11,12.The purpose of this review is to provide a summary of the evidence and compare the role of PET/CT or MRI in radiotherapy treatment planning for gastrointestinal cancers, mainly for anal, esophageal, rectal and pancreatic cancer, where the role of radiotherapy in the treatment strategy is more prominent ,14,15,16This systematic review was performed using structured search terms following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines .We searched PubMed databases with the search terms  OR  OR  OR (pancreatic cancer)) AND (MR OR MRI OR (magnetic resonance) OR (Positron Emission Tomography) OR (PET CT) OR PET) AND ((radiotherapy treatment planning) OR (interobserver variation) OR (interobserver variability) OR (delineation) OR (contouring) OR (target volume determination)). An additional search of the reference lists of the related articles was also performed. We assessed the full text to determine the eligibility of the articles. Studies were considered eligible if they fulfilled the following three criteria: (1) studies including patients with anal, esophageal, rectal or pancreatic cancer, (2) studies including PET/CT or MRI for radiotherapy treatment planning and (3) studies reporting interobserver variability or changes in treatment planning volume due to different imaging modalities or the correlation between the imaging modality and histopathological specimen. All the searches were limited to full-text articles and studies published in English.The search was conducted with a start date of the studies on 31 January 2003 to 31 January 2023. An initial search of the literature retrieved 1396 results, 1318 studies were excluded after reviewing the titles and abstracts. Seventy-eight were considered eligible for analysis, but forty-three were eliminated upon full-text review. We retrieved six articles from an additional search of the reference lists of related articles. Finally, the remaining 41 studies were included in the review .FDG-PET/CT for anal canal cancer (AC) has an overall sensitivity of 93% and a specificity of 76% . MoreoveIdentifying lymph node metastases (LNMs) is crucial for curative radiotherapy treatment planning in AC, especially in the context of adequate dose coverage of the involved nodes. Several studies have investigated the potential impact of FDG-PET/CT in target volume delineation in patients with AC who were candidates for curative radiotherapy. Mahmud et al. observed that treatment plans were modified in 12.5 to 59.3% of patients when FDG-PET/CT was used, which consisted mainly of a prescribed radiotherapy dose or field changes ,20. MoreOn the other hand, the elective coverage of regional lymph node stations in definitive radiotherapy treatment is also important. Omitting the treatment of possible microscopic, involved lymph nodes has been associated with a higher risk of local failure . There aRegarding primary tumor contouring, FDG-PET/CT was less sensitive than MRI in identifying T4 disease. According to the analysis, FDG-PET/CT could not provide the necessary anatomical detail required, and MRI remains the modality of choice for primary GTV contouring. FDG-PET/CT is sensitive in identifying the primary tumor but cannot fully characterize it . Anotherp = 0.053). They concluded that due to the rather good agreement, either modality can be used for the standard target delineation of anal cancer [Furthermore, studies assessing the interobserver variability in delineation between different imaging modalities are also important. Rusten et al. compared the target volume delineation of anal cancer using FDG-PET and MRI with respect to interobserver and inter-modality variability. It showed that GTV on FDG-PET had a lower interobserver variability in terms of Dice coefficients, i.e., 0.80 for FDG-PET and 0.74 for MRI, but they were not significantly different from each other  because of its ability to detect metastatic disease, including LNMs, with 66% sensitivity and 96% specificity . Both nep = 0.006), which was also confirmed using a multivariate analysis. The authors concluded that the use of FDG-PET/CT improved patients’ outcomes probably due to the more accurate staging and adequate coverage of the target volumes [Further studies analzsed the role of FDG-PET/CT in treatment planning for EC in comparison to CT regarding potential geographic misses. Jimenez et al. compared the target volumes and tumor lengths defined by fused FDG-PET/CT in comparison to CT simulation. The GTVnode was significantly greater on FDG-PET/CT. The Dice similarity coefficient analysis showed excellent agreement for GTVtumor, i.e., 0.72, but it was very low for GTVnode, i.e., 0.25. The study showed that CT simulation, without taking into account FDG-PET/CT information, could leave LNMs outside the radiotherapy treatment volume. Only 55.2% of patients had LMNs evident in both image data sets . Moreove volumes .On the other hand, Muijs et al. found no local recurrence following CT-based radiotherapy that could have been prevented by FDG-PET/CT. Ninety patients were planned for radiotherapy on the basis of CT simulation; all the patients also had FDG-PET/CT scans prior to radiotherapy. After treatment, the treatment volumes were adjusted based on the FDG-PET/CT when necessary. In 32 patients (36%), >5% of the PET CT-based GTV would have been missed if the treatment planning was CT-based. Local recurrences were seen in 10 patients (11%). There were three in-field recurrences, four regional recurrences outside both CT-based and FDG-PET/CT-based CTV and three recurrences at the anastomosis; none of these recurrences were considered preventable by FDG-PET/CT .p = 0.05), respectively [Another potentially important role for FDG-PET/CT in EC radiotherapy treatment planning is the determination of the primary tumor. Studies comparing the length of the tumor on preoperative FDG-PET/CT scans and on histopathologic specimens after surgery showed a good correlation ,43,44. Fectively . On the ectively . Nowee eectively .CT and GTVPET/CT were 0.39 ± 0.15 and 0.58 ± 0.10, respectively (p = 0.005) [p < 0.001). The intraobserver CIgen for senior radiation oncologists was larger than that for the junior radiation oncologists (p < 0.001). The use of multimodal imaging, including CT, endoscopy/EUS, esophagography and FDG-PET/CT for target delineation reduced the interobserver variability [However, in the case of cervical EC, Toya et al. observed a reduced interobserver variability for the delineation of GTV on FDG-PET/CT scans compared with CT images alone. The mean interobserver conformity index (CI) of the GTV= 0.005) . Moreoveiability .3D was contoured on a three-dimensional (3D) CT image without referencing the FDG-PET/CT image. The GTVPET-ref was contoured on the 3D-CT image while referencing the FDG-PET/CT image. The GTVPET-reg was contoured on images derived with the deformable registration of 3D-CT and FDG-PET/CT. No significant difference was found between the GTVs delineated based on visual referencing or deformable registration [A study conducted by Shi et al. showed that it is also feasible to delineate the GTV of primary thoracic EC with reference to the diagnostic FDG-PET/CT image in the absence of planning FDG-PET/CT images. The GTVstration .MRI is not yet an established method for radiotherapy treatment planning for EC, but it is promising because of its excellent soft tissue contrast; additionally, it is a non-invasive imaging modality. Hou et al. compared the GTV longitudinal length measured using different imaging modalities—CT, T2-weighted MRI (T2 MRI) and DWI—with the pathological lesion length to determine the most accurate imaging modality. They concluded that DWI correlated with pathological lesion lengths more precisely compared to CT or T2 MRI. DWI scans fused with CT images can be used to improve the accuracy of GTV delineation in EC .Wollenbrock et al. evaluated the feasibility of target delineation on T2 MRI and T2 MRI combined with DWI (T2 MRI+DWI) compared with FDG-PET/CT. No differences were observed in CIgen . The most variation was seen at the cranial–caudal borders, and the addition of DWI to T2 MRI can reduce the variation in the caudal border delineation in gastro-esophageal junction tumors. The study showed that MRI-based GTV delineation of the EC is feasible, with an interobserver variability comparable to that in FDG-PET/CT; a potential pitfall could be a lack of experience in delineation using MRI .Wang et al. investigated the assessment of the primary tumor and regional lymph nodes using FDG-PET/MRI, FDG-PET/CT, MRI and contrast-enhanced CT (CECT). The pathology specimen was used as a reference standard to assess the accuracy of all the imaging modalities. For primary tumor staging, the accuracy of PET/MRI, MRI and CECT in comparison to the pathological specimen was 85.7%, 77.1% and 51.4%, respectively. For lymph node assessment, the accuracy of PET/MRI, PET/CT, MRI and CECT was 96.2%, 92.0%, 86.8% and 86.3%, respectively. 18F-FDG PET/MRI has advantages over 18F-FDG PET/CT, MRI and CECT in the preoperative assessment of primary tumors and regional lymph nodes of EC .FDG-PET/CT seems superior, especially in the detection of LNM in EC; consequently, it is essential when determining CTV as in the elective or involved-field irradiation. The role in determining the primary tumor is not so clear; apparently, FDG-PET/CT is suitable for localizing the tumor. MRI could play a role in GTV determination, but there are insufficient data to date .Surgery is the cornerstone treatment for rectal cancer (RC), but organ-preserving strategies (watchful waiting) are gaining importance. Total neoadjuvant therapy (TNT) is a reference treatment in locally advanced RC with high-risk criteria, including extramural vascular invasion or the involvement of the mesorectal fascia, but it is also used in the treatment of other tumors with high risk criteria such as T4 tumors or positive lymph nodes on imaging . AccuratSeveral papers explored the role of MRI in treatment planning for RC. One of the first papers by O’Neill et al. reviewed imaging and planning data for patients with locally advanced low RC. Eligible patients were defined using MRI as having cT3 tumors deemed to require abdominoperineal excision or cT4 and cT3 tumors with involved mesorectal fascia. Tumor volumes and location were compared using sagittal pre-treatment MRI and planning CT. MR-based rectal tumor volumes were smaller and thus lay further from the anal verge, facilitating the relative sparing of the anal sphincter . Tan et The role of FDG-PET/CT in treatment planning for RC has also been analyzed. In a study, they included patients with cT2-4N0-2M0 tumors as well as less advanced stages in comparison to the previously mentioned papers. The GTV volumes based on FDG-PET were significantly smaller , which wThe same study showed that in up to 29% of patients, the CTV based on FDG-PET extended outside the CTV used in clinical practice . HoweverSeveral studies investigated interobserver variability for different imaging modalities in order to achieve better agreement in delineation for RC between radiation oncologists. Two studies compared the GTV delineated on T2 MRI and DWI images to assess whether the agreement was improved by DWI. The results showed a smaller target volume for DWI, which did not translate into a better agreement ,61. BurbPET/CT was significantly smaller than the mean GTVCT. FDG-PET/CT significantly increased the interobserver concordance index in contouring GTV compared with CT-only-based contouring: 0.56 versus 0.38 (p < 0.001). The study suggests that PET CT could reduce interobserver variability when contouring boost volumes [p = 0.009). DWI resulted in a smaller volume of delineation compared to the other image modalities. They concluded that DWI could be an optimal strategy for boost volume delineation for dose escalation in patients with RC [FDG-PET/CT was also assessed in studies analyzing interobserver variability for the delineation process in RC, where the GTV was contoured using CT and FDG-PET/CT sequences. Of the 30 evaluable preoperative patients, the mean GTV volumes . Similar volumes . Recentl with RC . AccordiTo sum up, the presented studies showed that MRI could have a role especially in determining the tumor boundaries in the anorectal and sigmoid region. DWI is gaining importance and could have a role in sub-volume treatment planning strategies as it represents the area of greatest diffusion restriction, which could be potentially radioresistant. The addition of FDG-PET/CT could add other important information to the final target volume, could help in tailoring the cranio-caudal border of the tumor and could reduce interobserver variability. Its role in CTV delineation remains investigational .The role of radiotherapy in pancreatic cancer (PC) is still controversial. There is a lack of robust evidence, but some patients may still benefit from local treatment. Radiotherapy can be delivered in the neoadjuvant setting for borderline resectable or up-front unresectable tumors. The optimal imaging modality to accurately define the GTV for radiotherapy of pancreatic cancer is unknown. There were only scarce data retrieved from our analysis. Dalah et al. explored the potential role of using various imaging modalities including MR, FDG-PET/CT and CT to define the treatment targets for radiation therapy of PC. Patients were classified into three categories according to whether the tumor was surgically resectable, borderline resectable or locally advanced. Significant differences were found between the volumes of GTV contoured using several modalities. The authors observed the ability of metabolic imaging of FDG-PET to accurately define the GTV. They discussed the accuracy of FDG-PET, which is worse in assessing tumors larger than 4 cm, which is partially due to the low metabolic rates in portions of larger tumors. However, for hypermetabolic tumors smaller than 2 cm, the sensitivity of FDG-PET is superior to that achieved with CT, meaning that FDG-PET could be appropriate for treatment planning for resectable or borderline tumors. The OAR volumes based on MRI are generally smaller than those based on CT. They suggested that further imaging studies with pathological correlation are required to establish an optimal imaging modality for PC .Li et al. compared the differences between CE FDG-PET/CT and CECT in target volume delineation in the treatment plan for locally advanced pancreatic cancer (LAPC). Twenty-one consecutive patients with LAPC underwent both non-CECT and FDG-PET scans; eleven of them also underwent CECT scans. The GTVs were smaller when contoured on FDG-PET/CT scans in comparison to CECT or non-CECT. The co-registration of FDG-PET/CT with CECT could improve the accuracy of GTV delineation in LAPC and could reduce the adverse effect of irradiation .In another study, the role of FDG-PET/CT in the delineation process for unresectable locally advanced PC (LAPC) in comparison to CT was also analyzed. Changes in GTV delineation were necessary in five patients based on FDG-PET/CT information. In these patients, the average increase in GTV was 29.7% due to the inclusion of additional LNMs and extension of the primary tumor beyond that defined by CT. Therefore, the co-registration of FDG-PET and CT images in unresectable LAPC can improve the delineation of GTV and theoretically reduce the likelihood of geographical misses .Liermann at al. investigated a novel tracer, FAPI, in tumor volume determination in LAPC. The GTVs of seven patients with LAPC were contoured by six radiation oncologists. Additionally, FAPI-PET/CT was used to automatically delineate the GTV. There was no significant difference between the volumes of automatic FAPI-GTVs and most of the GTVs manually contoured by radiation oncologists. They concluded that FAPI-PET/CT can be used as an additional imaging modality to improve decision-making in target definition .Functional imaging with FDG-PET or other tracers could have a role in target volume determination for LAPC, but further investigations are warranted .Accurate tumor delineation is necessary as precision radiotherapy is becoming important in the treatment strategies for gastrointestinal cancers, especially anal, esophageal, rectal and pancreatic cancer.FDG-PET/CT seems especially essential for LNM delineation and the definition of the elective nodal irradiation in AC and EC due to its accuracy. The addition of FDG-PET/CT modifies the CTV and could potentially avoid a geographic miss and therefore allow for smaller treatment fields without risking undertreatment. FDG-PET/CT helps to define the regional lymph node stations at greater risk of micrometastasis and thus the elective treatment volume in definitive chemoradiotherapy. Its role in primary tumor delineation for EC is not as clear. It seems that FDG-PET/CT is appropriate for tumor localization but not for defining precise boundaries, for which another imaging modality could be more adequate, for instance MRI, but there is not sufficient evidence.To date, it seems that MRI has an important role in determining the primary tumor, especially in the pelvis, for the delineation of AC and RC. MRI for GTV tumor delineation in AC could reduce the target volume and consequently reduce the dose to OAR. Similarly, MRI has an important role in tumor target delineation in RC, where it can help to define the GTV in the anorectal and sigmoid region. FDG-PET/CT for GTV determination in RC could tailor the craniocaudal border delineation and improve interobserver agreement.It appears that especially DWI sequences are becoming increasingly important in boost treatment planning strategies. DWI enables GTV definition with a high level of precision and allows dose escalation to the area of the greatest restriction of diffusion.Interestingly, in our search, we retrieved fewer articles regarding PET/CT and MRI for target volume determination in RC in comparison to EC. One of the reasons could be that the reference treatment for locally advanced RC is still neoadjuvant therapy  followed by surgery, so the focus of the studies is on the optimal treatment strategy rather than imaging for target volume delineation. In addition, MRI is the gold standard for diagnostic imaging in RC, and probably the scientific community does not feel the need to perform studies comparing MR and CT in this setting. Moreover, dose escalation for LNM in RC is not a routinely adopted strategy in standard treatment protocols. However, as organ sparing in RC is gaining importance, dose escalation to LNMs could be a future strategy, and new studies are needed.Target definition in PC is challenging due to the anatomical and physiological specificity of the region—sometimes it is difficult to distinguish the tumor from fibrosis in PC. MRI and FDG-PET/CT could improve target volume delineation in PC, but to date, there is insufficient data. The use of other tracers is still investigational.In conclusion, both PET/CT and MRI are gaining an important role in radiotherapy target volume determination for gastrointestinal cancers, for the delineation of the primary tumor, pathological lymph nodes and elective volume definition. Further studies to determine the optimal combination of imaging modalities for radiotherapy treatment planning are warranted. Whenever possible, studies with pathohistological confirmation should be performed, as they represent the real correlation between the tumor and different imaging modalities. Research on interobserver variability with different imaging modalities is also recommended, especially with a dosimetric analysis.


P < 0.004) and parents’ psychiatric problems . Among females, conduct disorder , being a bully/bully-victim  and parental substance-use problems  were related to heavy BZD use. The mean (±SD) age of first BZD prescription was significantly lower in heavy, compared with mild users . Heavy, compared with mild, BZD use is associated with female suicide attempts . Physicians should be cautious when prescribing BZDs to young adults and must allocate treatment to those with carefully evaluated clinical indications.We investigated factors associated with benzodiazepine (BZD) use during late adolescence and early adulthood. The study population consisted of 508 adolescents admitted to psychiatric inpatient care between April 2001 and March 2006. Information on adolescents’ family- and school-related factors, suicidality and psychiatric disorders were obtained by semistructured interviews. Data on BZD prescriptions from 1999 to 2012 were collected from the Social Insurance Institution of Finland. In males heavy BZD use associated with adolescent substance-use disorder (OR, 3.5;  Benzodiazepines (BZDs) are a group of drugs that bind allosterically to specific receptors on gamma-aminobutyric acid chloride channel and potentiate the inhibitory effects of the channel. Consequently, BZDs reduce the amount of action potentials and turnover of several neurotransmitters . BZDs haThe potential adverse effects of BZDs, such as daytime drowsiness, cognitive decline and deficiency in balance resulting falls, are well known . Together with opioids, BZDs are the most commonly abused prescription drugs . TherefoThe aim of the present study is to investigate the relation of adolescence-related psychosocial factors (i.e. family- and school-related characteristics), suicidality, nonsuicidal self-injury (NSSI) and adolescent psychiatric disorders to participant’s prescribed BZDs during late adolescence or early adulthood. For this purpose, we utilized a population-based data of former adolescent psychiatric inpatients from Northern Finland. We focused on BZDs typically used to treat daytime anxiety  and clonazepam, due to their potential risk for misuse, particularly among young adults  who were admitted to the inpatient adolescent psychiatric ward at Oulu University Hospital between April 2001 and March 2006 . The ward provides treatment for all adolescents from the area of Northern Finland (the provinces of Oulu and Lapland) requiring acute psychiatric hospitalization. The mean age of the study participants on admission was 15.5 years (SD = 1.3). The study was approved by the Ethics Committee of Oulu University hospital.This study is part of clinical follow-up project, which was initiated to examine the association of various psychosocial risk factors to the outcomes of severe psychiatric and substance-use disorders among adolescents. The initial study population consisted of 508 adolescents , or they had only a single or occasional prescriptions (n = 44) of BZDs during the follow-up. Thus, the final study population analyzed in this study comprised 445 former adolescent psychiatric inpatients .Regarding the central focus of our study, we excluded 63 study participants, because their BZD prescriptions were probably due to the presence of an epilepsy diagnosis , was used to gather information about the home environment at admission, parental characteristics and school-related factors.The Schedule for Affective Disorders and Schizophrenia for School-Aged Children (K-SADS) to obtain Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) diagnoses. K-SADS-PL is shown to be reliable method for defining DSM-IV psychiatric disorders in children and adolescents  of the study participants was obtained from the Social Insurance Institution (SII) of Finland. The data regarding prescriptions were available up to the end of 2012  when the age of participants varied between 19 and 30 years. The age (in years) at first prescription of BZD, as well as the total number of prescriptions of BZDs, was calculated for each study participant.We categorized the subjects with BZD prescriptions into two subgroups based on the amount and the total cost (in euros) of BZD purchases:(1). ‘Mild users’ had nine or less prescriptions or the total amount of money spent on BZDs during the follow-up time was 50 euros or less.(2). ‘Heavy users’ had either 9 or more prescriptions or had spent 50 euros or more on purchases.n = 70 heavy users and n = 38 mild users). The control group (n = 337) comprised participants without any BZD purchases.The reason we took into consideration the money spent on BZDs, in addition to the amount of medicine acquired, was the differences in costs between different BZDs. The total number of BZD users in this study was 108  two-parent family , (b) single-parent family, and (c) out-of-home placement .Information on parental employment status  and problems related to parental mental health and substance-use was obtained from the EuropASI. Parental mental health and substance-use problems were based on each adolescent’s self-report as to whether they perceived that their mother or father would have required treatment for psychiatric (yes/no) or substance use  problems (yes/no).et al., 2011). The following three categories for bullying behavior was formed: (a) no involvement in bullying behavior, (b) victim of bullying, (c) bully/bully-victim (participants being bullies or both bullies and victims of bullying).Information on bullying behavior was obtained from the K-SADS-PL. In the School Adaptation and Social Relations section of the interview, adolescents were asked whether they had ever been victims of bullying. Information on the bullying behavior was based on screening items regarding conduct disorder. The adolescents were asked: ‘Has there ever been a time when any kids really got on your nerves? Did you sometimes do things to get back at them ? How often did you do these things?’ Bullying was regarded as being present if the subjects had threatened or intimidated another on three or more occasions  or received additional help  at school (yes vs. no).Information on an adolescent’s current suicidality  and NSSI was based on the K-SADS-PL’s section on depressive disorder. Suicidal ideation was defined as being present when thoughts of suicide had been recurrent, and the adolescent had also considered the method of suicide. The information on suicide attempts, their seriousness and medical lethality was retrieved from two questions from the K-SADS-PL: (a) ‘have you actually tried to kill yourself?’, and (b) ‘how close were you to dying after your most serious suicidal act?’ The ‘suicide attempts’ group consisted of those adolescents who were at threshold level in either of the two items for suicidality. In terms of suicidality, study participants were categorized into three subgroups: (a) no suicidal behavior, (b) suicidal ideation and (c) an attempted suicide. Additionally, the NSSI was defined to be present (yes/no) if an adolescent had frequently carried out physical self-harming acts (>4 times a year).The DSM-IV psychiatric disorders were assessed using the K-SADS-PL interview. These disorders were classified into five major psychiatric diagnostic groups, as follows: psychotic disorders , anxiety disorders , affective disorders , conduct disorders including ADHD  and substance-use disorders . An adolescent may have had diagnoses belonging to multiple categories of psychiatric disorders.The statistical significance of group differences was assessed with Pearson’s Chi-square or Fisher’s Exact test. A logistic regression analysis was used to examine the association of psychiatric disorders, diagnosed in adolescence, to the likelihood of BZD use by early adulthood, after controlling for adolescence-related covariates . When calculating the age at first BZD prescription, one single prescription of BZD for one study participant was left out from our analysis as it was deemed a potential outlier, because it was occurred at 4 years of age. The statistical software used in analyses was IBM SPSS statistics, version 27 .n = 445), the use of BZDs was found in 108 (24.3%) study participants, the proportions being 53 (28.2%) male (n = 188) and 55 (21.4%) female (n = 257) participants. In male participants with BZDs, 16 (30.2%) were mild and 37 (69.8%) heavy users of BZDs, whereas in female participants 22 (40.0%) were mild and 33 (60.0%) heavy users of BZDs.In our total sample of former adolescent inpatients . There was no statistically significant sex difference in age of first BZD prescription either among mild (P = 0.905) or heavy BZD users (P = 568).As Fig. P < 0.001), family structure being less commonly two-parent family  but more commonly out-of-home placement , parents with psychiatric problems , and they had substance-use disorder diagnosed during index hospitalization in adolescence . Analyses within male BZD users (Table P = 0.031).Table P < 0.001], parental psychiatric problems  and substance-use disorder in adolescence . Similarly, heavy use of BZDs, compared with controls, was related to higher age at admission , parental psychiatric problems  and substance-use disorder in adolescence . Further, a decreased likelihood for suicide attempts in adolescence was observed  when comparing heavy BZD users with mild users. In comparison of mild users of BZD with controls (data not reported in Table P = 0.008) was associated with mild BZD use.When all adolescence-related characteristics of male participants (presented in Table P = 0.010), their family structure was less commonly two-parent family  but more commonly out-of-home placement , parents had substance-use problems , they less commonly belong to the group of no bullying behavior  but more commonly were bullies/bully-victims , they have received special services  and repeated grades  in school, and they were diagnosed with anxiety  and conduct  disorders during index hospitalization in adolescence. Within female BZD users, the heavy users differed from mild users with regard to bullying behavior , previous suicide attempts , and diagnosis for conduct  and substance-use  disorder.As seen in Table P = 0.008), parental substance-use problems , being a bully/bully-victim  and having had special services in school . Further, heavy use of BZDs in female participants, compared with controls, was related to higher age at admission , parental substance-use problems , being bully/bully-victim  and being diagnosed with conduct disorder during index hospitalization in adolescence . In comparison between heavy users and mild users, significant predictors for heavy use of BZDs were suicide attempts , and a diagnosis of conduct  and substance-use  disorder in adolescence. None of the adolescence-related characteristics in females were associated with mild use of BZDs, compared with controls.In the assessment of statistically significant adolescence-related predictors for use of BZDs among female participants Table , the sigOur study of former adolescent psychiatric inpatients showed that adolescent substance-use disorder, in both sexes, and conduct disorder among females, was related to heavy BZD use later in life. Suicide attempts in adolescence were associated with heavy BZD use in females but, interestingly, heavy BZD use was not associated with anxiety-, mood- or psychotic disorders, where short-term BZD use may be clinically justified.Our study showed that the younger the age at first BZD prescription, the more likely it was a participant became a heavy BZD user, both in male and female subjects. This finding is in line with earlier studies  and, therefore, all BZD purchases as prescription medication are captured from records of SII. However, we were not able to calculate the defined-daily-doses, as our data did not include information on the strengths or different package sizes of BZDs. An important observation is that our results are likely to underestimate the true levels BZD use, because medicines may also have been illegally purchased on the black market. Our study population represents adolescents in need of psychiatric inpatient treatment and, thus, our findings cannot directly be generalized to the whole Finnish adolescent population. We focused on adolescence-related factors in terms of later use of BZDs, which evidently calls for further research to examine these associations in terms of psychiatric morbidity after adolescent index hospitalization.Our findings suggest that BZD prescriptions among adolescents and young adults may not always be based on an adequate psychiatric assessment and appropriate clinical indications. In particular, the high levels of BZD use among participants with a history of adolescent substance-use disorder are an alarming finding. Adolescents with suspected substance-use problems need careful assessment, case management and age-specific treatment arranged in appropriate substance-use or other psychiatric services targeted to adolescents. A treatment plan should be devised cooperatively, with parents and/or significant others involved in all stages of an adolescent’s treatment program. Clinicians should be cautious when treating adolescents and young adults who are repeatedly seeking BZD prescriptions and should have a lower threshold for making referrals for diagnosis-based psychosocial interventions, including substance-use treatment.There are no conflicts of interest.