To describe the use of tramadol and other analgesics in Denmark focusing on the impact of media attention (June and December 2017) and regulatory actions (September 2017 and January 2018) on the use of tramadol.Using nationwide registries, we identified all adults who filled a prescription for tramadol and other analgesics from 2014 to 2019. We described incidence rates, prevalence proportions, and total use of tramadol and other analgesics over time. We also described switching between analgesics, treatment duration, skewness in drug use, and doctor-shopping.From early 2017 until the end of 2019, total tramadol use decreased markedly while the use of morphine and oxycodone decreased slightly. The quarterly prevalence of tramadol use decreased from 32/1000 individuals in 2014 to 18/1000 at the end of 2019, dropping mainly at the time of media attention. Concomitantly, the quarterly prevalence increased for oxycodone (from 5.1 to 8.2) and morphine (from 8.5 to 9.8), mainly due to more short-term and sporadic users, and decreased for codeine (14 to 9.6). From 2014 to mid-2017, the incidence of tramadol use was stable (around 2.2/1000 person-months) but dropped in June 2017 to 1.7/1000, coinciding with the media attention. The incidence of tramadol use continued to decrease (to 1.1/1000 at the end of 2019).We identified a decline in tramadol use coinciding with the media attention in 2017 and continuing during regulatory actions. There was generally no evidence of unintended effects on the utilization of opioids related to the media attention and regulatory actions.The online version of this article (10.1007/s00228-020-03016-6) contains supplementary material, which is available to authorized users. The use of prescription opioids, including tramadol, has increased worldwide during the last decades resulting in a dramatic increase in overdoses and opioid use disorders, especially in the USA –3. In DeTramadol is a centrally acting synthetic opioid with monoaminergic actions, and both abuse and addiction have been reported in several previous studies , 7. DespAt the same time, there has been an increased awareness by the Danish Health Authority on the increasing use of opioids, including tramadol. Since 2007, several initiatives have been launched including guidelines and educational courses directed at Danish physicians , 14. In Previous studies from other countries have observed changes in drug utilization following regulatory actions and media attention –19. CurrIn this descriptive nationwide drug utilization study, we used the Danish health care registries to describe the use of tramadol and other analgesics including other opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), gabapentinoids, tricyclic antidepressants (TCAs), and serotonin and norepinephrine reuptake inhibitors (SNRIs) in adults from 2014 to 2019.The Danish National Prescription Registry contains individual-level information on all prescription drugs dispensed at Danish outpatient pharmacies since 1995 . PrescriData on hospital diagnoses was obtained from the Danish National Patient Register  and we uThe data sources were linked by a unique identifier (CPR-number), which is assigned to all Danish residents at birth or immigration.All drug (ATC codes) and diagnosis codes ) used in this study are provided in Online resource 0 codes) The study cohort included all adults (≥ 18 years) living in Denmark from January 1, 2014, to December 31, 2019. For this cohort, we identified all filled prescriptions for tramadol, other opioids, NSAIDs, gabapentinoids , TCAs, and SNRIs. We included the use of NSAIDs and gabapentinoids, to assess possible changes in utilization patterns of other non-opioid analgesics, and TCAs and SNRIs to assess switching between analgesics. We differentiated between new users and prevalent users. New users were defined as individuals redeeming a prescription with no prior prescription for the same drug or drug class within the last 5 years.Opioids included all opioids available for oral or transdermal use . We categorized opioids according to the four most frequently used individual opioids in Denmark: morphine, oxycodone, tramadol, and codeine. We included a category with “other opioids,” which included the remaining opioids in the ATC group for opioids (N02A*), which, when considering volumes sold, mainly comprised transdermal opioids. In Denmark, three combination products of codeine are available, and these were all included in the codeine category. In order to restrict the analysis to drugs used to treat pain, we only included codeine tablets .We used oral morphine equivalents (OMEQs) to quantify the equipotent amount of opioids used and applied conversion factors recently published by Svendsen et al. and Jarlbaek , 27. ConTime of media attention was determined by the first documentary broadcasted in June 2017 and supported by a Google Trends query showing a large search activity of “Tramadol” in Denmark during June 2017 , 28.We divided the analysis into seven parts where we described the use of tramadol and other analgesics during 2014–2019 and described changes in treatment patterns over time by comparing three study years, i.e., 2016 (before media attention and regulatory actions), 2017 (during media attention and regulatory actions), and 2019 (after media attention and regulatory actions).First, we described users of tramadol and other opioids according to sex, age, and morbidities. This was done separately for users in 2016, 2017, and 2019. Morbidities were assessed as hospital diagnoses recorded in the Patient Registry at any time prior to January 1 of the year being analyzed.Second, three separate analyses were performed to describe changes in analgesic use over time from January 1, 2014, to December 31, 2019. These included (A) total drug use per month, calculated by dividing the amount of dispensed milligram OMEQs  by the number of adults living in Denmark on the 1st day in each quarter; (B) yearly and quarterly prevalence proportions of use of tramadol, other opioids, NSAIDs, and gabapentinoids, calculated for all adults living in Denmark on January 1 for each year or, for the quarterly prevalence, 1st day in each quarter in the denominator; and finally, (C) monthly incidence rates calculated by dividing the monthly number of new users (as defined above) by the total number of adults in Denmark on the 1st day in each quarter.Third, to assess possible switches between analgesics in relation to the use of tramadol, we calculated the proportion of new tramadol users in 2016, 2017, and 2019 who had filled other analgesics  during the previous 3 months or during the 3 months according to the first tramadol prescription.Fourth, as a crude measure of treatment duration, we calculated the proportion of new tramadol users who redeemed 1, 2, or ≥ 3 tramadol prescriptions in the year following their first tramadol prescription. The analysis was performed separately for 2016, 2017, and 2018 (to ensure a full year of follow-up). The crude measure of duration was applied due to preliminary analyses showing a very low number of long-term users, rendering “drug survival” analyses less useful .Fifth, we investigated the medical specialty of prescribers issuing prescriptions for tramadol. We calculated the proportion of incident tramadol prescriptions and prevalent tramadol prescriptions (defined as non-incident) issued by different types of prescribers in 2016, 2017, 2018, and 2019. The type of prescriber was classified into six categories including (1) general practitioners (GPs) , (2) private practicing specialists, (3) hospital prescribers, (4) dentists, (5) others, and 6) missing.Sixth, we described the skewness of tramadol use by generating Lorenz curves for the years 2016, 2017, and 2019. Lorenz curves describe skewness in the use of a drug on a population level and have been described in detail elsewhere . It may Finally, to assess the occurrence of doctor shopping among users of tramadol, codeine, oxycodone, and morphine, we calculated the proportion of individuals with 1, 2, 3, 4, or ≥ 5 different unique prescribers in 2016, 2017, 2018, or 2019. To ensure a relevant population, this analysis was restricted to individuals redeeming ≥ 5 prescriptions of the given drug during the given year. In Denmark, 98% of the population are assigned to a specific GP practice which acts as a gate-keeper to specialist treatment . The numAll analyses and graphics were performed using Stata Release 15.2 .Approval from the Ethics Committee was not required according to Danish law . In termWe identified 1,328,568 unique users of opioids of which 63% had redeemed one or more prescription for tramadol between January 1, 2014, and December 31, 2019. In 2019, 247,335 (59%) of opioid users were female and the median age was 63  years  and the total tramadol use decreased . Concomitantly, the total use of oxycodone  and morphine  decreased slightly  but dropped in June 2017 to 1.7/1000 person-months coinciding with the media attention and further to 1.6/1000 person-months and 1.4/1000 person-months in September 2017 and January 2018, respectively Fig. . At the The proportion of new tramadol users in 2019 who had filled a prescription for NSAIDs, TCAs, SNRIs, gabapentinoids, or another opioid within 3 months before initiating tramadol was 22%, 0.89%, 1.8%, 3.8%, and 7.9%, respectively, while the proportion of new tramadol users who in the subsequent 3 months filled a prescription for these drugs were 34%, 1.5%, 2.0%, 8.4%, and 16%, respectively. The same patterns were observed in the years 2016, 2017, and 2018 (data not shown). An exploratory post hoc analysis revealed that in 2014, 33% of individuals who initiated morphine or oxycodone had used tramadol in the 3 months leading up to them initiating morphine/oxycodone. In 2019, this proportion was reduced to 16%.The proportion of new tramadol users who filled only one tramadol prescription increased from 63% in 2016 to 73% in 2018. Furthermore, the proportion who filled 2 or ≥ 3 prescriptions in 2016 was 17% and 21%, respectively, and the corresponding numbers were 15% and 12% in 2018, respectively.Tramadol was primarily initiated by GPs in 2019: 62%) and hospital prescribers (in 2019: 26%). This changed slightly over time from 2016 to 2019, with a slight decrease in the proportion of new treatments initiated by GPs (from 68 to 62%). Considering prevalent prescriptions, tramadol treatment was mainly maintained by GPs (in 2019: 92%) with no variation over time  or two prescribers (in 2019: 22%). The proportion of tramadol users who had tramadol prescribed by ≥ 5 prescribers declined from 0.74% in 2016 to 0.29% in 2019. Corresponding results are available for other opioids in Online resource 019: 72% In this nationwide study of the use of tramadol and other analgesics, we found a considerable decrease in both incidence, prevalence, and total use of tramadol coinciding with the media attention in June 2017, a decline which persisted during the regulatory actions. In the same period, a slight increase was noted in the number of new users of morphine and oxycodone, however, with a slight decline in the total use of these drugs, indicating a larger proportion of short-term or sporadic users. Further, the use of gabapentinoids increased and that of NSAIDs decreased during the study period. Tramadol was mainly initiated by GPs as well as hospital prescribers, while continued use was almost exclusively prescribed by GPs. The proportion of heavy or long-term tramadol users was low, while sporadic or short-term tramadol use was more common, which warrants further investigation. However, the skewed distribution of tramadol use was consistent over time, i.e., seemingly not a result of media attention or regulatory actions. There was very limited and diminishing evidence of doctor-shopping for tramadol. We identified a periodical rhythm in both the prevalence and incidence of codeine, which might reflect use of codeine tablets as cough suppressants.The principle strength of the study is the use of nationwide registries of high quality , 24, 25,Media attention affecting use of drug therapy has been described numerous times. An example is the debate on the benefit:risk ratio of statins following publications of several articles in The BMJ , 35, wheInterruptions in prescribing patterns, e.g., due to media attention or regulatory actions, can have unforeseen and unwanted consequences. As an example, Chen et al. found an increase in the use of selective serotonin reuptake inhibitors (SSRIs), benzodiazepines (BZDs), and SNRIs after the reclassification of tramadol in the UK . While oWe identified a decline in the use of tramadol coinciding in time with the media attention in 2017. This decline continued during the regulatory risk minimization actions taken in 2017–2018. During the same period, the use of other opioids decreased slightly. There was generally no evidence of unintended effects on the utilization of opioids related to the media attention and regulatory actions. In total, this provides strong evidence for the combined effect of media attention and regulatory actions in changing prescribing practices within a therapeutic area which is currently much debated.ESM 1(DOCX 25 kb)ESM 2(DOCX 329 kb)


Multiple myeloma (MM) is a plasma cell disorder that is on the rise throughout the world, especially in the US, Australia, and Western Europe. In the US, MM accounts for almost 2% of cancer diagnoses and over 2% of cancer deaths . Incidence has risen by 126% globally and over 40% in the US since 1990, while global mortality has risen by 94% and US mortality has fallen by 18%. The 5 year survival in the US has more than doubled over the past decades with the introduction of new targeted therapies and transplant techniques. Risk factors for MM include age (average age of diagnosis is 69), race (African Americans are over double as likely to be diagnosed), sex (men are at a 1.5× risk), and family history. Diagnosis includes serum or urine electrophoresis and free light-chain assay but requires bone marrow biopsy. It is distinguished from smoldering myeloma and monoclonal gammopathy of undetermined significance by a high (>3 g/dL) level of M-protein  and the presence of CRAB  symptoms, which include hypercalcemia, renal failure, anemia, and bone pain, suggesting an end-organ damage. International staging system staging involves beta 2 microglobulin and albumin levels, while the revised system considers prognostic factors such as lactate dehydrogenase levels and chromosomal abnormalities. Front-line management includes induction regimen, maintenance therapy and hematopoietic cell transplantation for eligible patients and bisphosphonates or bone-stimulating agents for the prevention of skeletal events. Treatment for relapsed disease includes newly approved monoclonal antibodies like the CD38-targeting daratumumab, proteasome inhibitors, immunomodulating agents, and investigational therapies such as B cell maturation antigen Chimeric antigen receptor T cells. Multiple myeloma (MM) is a malignancy of plasma cells which is rising in incidence in the developed world. Plasma cells are mature antibody-producing B cells which reside in the bone marrow and are essential for maintaining humoral immunity. Multiple myeloma is characterized by a monoclonal proliferation of plasma cells resulting in the production of monoclonal antibody and end-organ damage . This caPlasma cell dyscrasias are classified based on the occurrence of these symptoms and levels of monoclonal antibodies . Those with the proliferation of plasma cells in their bone marrow and high antibody levels (M-protein), but without CRAB symptoms, are classified as smoldering myeloma (SM) . Those wAccording to the latest Global Cancer Observatory (GLOBOCAN) statistics, there were an estimated 160,000 cases of MM globally in 2018, accounting for 0.9% of all cancer diagnoses  7]. App. App7]. It is more common, and rising in incidence, in the developed world with the highest incidence in Australia, Western Europe, and the US. In the US, an estimated 32,000 cases were estimated to be diagnosed in 2020, accounting for 1.8% of all cancer diagnoses. This makes multiple myeloma the 14th most common neoplasm. The current estimated incidence rate, 7.0/100,000, is a 143% increase since 1975, when the incidence was 4.9/100,000 . App. App7]. In the US, 12,800 people were estimated to perish in 2020 from MM, accounting for 2.1% of all cancer deaths. While incidence has risen over the past decades, mortality has fallen due to the drastically increased survival. The current mortality of 3.3/100,000 is 18% below the peak of 4.0/100,000 in 1994. This reflects a 2.27× increase in 5 year survival over the past decades, from 23.7% in 1976 to 53.9% in 2016 .Survival is somewhat dependent on the stage at diagnosis, with a 74.8% 5 year survival for those with localized disease  and a 52.9% for systemic MM (the remaining 95% of diagnoses) . Atkin eMultiple myeloma is a neoplasm of older adults with the median age at diagnosis being 69 in the US. Over 60% of diagnoses are made in those greater than 65, and less than 15% of diagnoses are made in those under 55  11]. Th. Th11]. It is about 1.5× more common among men than women, globally. Suggested underlying factors include discrepancies in health-risk behaviors, such as smoking and alcohol consumption, and higher rates of obesity among men, though none of these risk factors have been proven in MM .It is more than twice as common among African Americans with an incidence of 16.5/100,000 among African American men and 12.0/100,000 among African American women . This discrepancy is over 3× among those below 50, showing that African Americans have a younger onset of disease, on average . Asians While family clusters of MM have been described, underlying predisposing germline mutations have yet to be characterized. Retrospective results from the Multiple Myeloma Consortium found that the odds ratio (OR) for MM among first-degree relatives was 1.90, (95% CI: 1.26–2.87) with an especially strong association among men  and African Americans  [Patients often present with CRAB symptoms. When MM is suspected, blood and urine electrophoresis should be performed to look for the monoclonal light-chain secreted by the neoplasm. Blood levels of IgG, IgM, and IgA can identify the isoform of light-chain produced. If levels are elevated but under 3 g/dL, the disease can be classified as SM or MGUS instead of MM. A serum-free light-chain (FLC) assay is more sensitive than urine . Serum albumin and beta-2 microglobulin (B2M) from bloodwork are also valuable for diagnosing and staging the disease. The definitive diagnosis requires a bone marrow biopsy with greater than 10% clonal plasma cells, or the presence of a plasmacytoma elsewhere. End-organ damage is necessary to distinguish from SM. Renal indices should be evaluated to a glomerular filtration rate, usually calculated by creatinine levels, that can be used to establish renal insufficiency. Imaging, such as CT , MRI, and PET scans, is used to uncover lytic bone lesions. If patients are unable to undergo these imaging procedures, a skeletal survey is used instead. Eighty percent of patients have some skeletal lesions, fractures, or osteopenia at the time of diagnosis ,19. The The two main staging systems used for MM are the international staging system (ISS) and the Durie–Salmon system (DSS). The ISS stratifies cases into three stages: Stage I for those with a B2M less than 3.5 mg/L and albumin > 3.5 g/dL, Stage III for those with a B2M greater than 5.5 mg/L, and Stage II for all those in between . The revWith the advent of new therapies, the overall survival of the disease has increased. With improving survival, there was a need for re-staging the disease for early diagnosis, treatment, and preventing end-organ damage . Until rBoth the ISS and DSS systems assess the tumor burden, but neither ISS nor DSS takes into consideration the biology of the disease, which determines the overall survival ,21,22.The revised international staging system (RISS) combines elements of tumor burden (ISS) and disease biology . It was The survival has more than doubled over the past decades due to the introduction of new chemotherapy combinations, targeted small molecule inhibitors, and monoclonal antibodies. In The results of the SWOG S0777 trial showed that the triplet regimen VRd  improved response rates, depth of response, progression-free survival, and overall survival compared with the then approved front-line regimen Rd  making a three-drug regimen the main stay of initial therapy for most patients with MM . This inFor most patients with standard-risk MM, the most commonly used induction therapy is VRd. VCd also called CyBorD  is an acceptable alternative for patients who are taught to have an increased risk of complication from the use of lenalidomide  . Other tIf patients relapse within 12 months of completion of initial therapy, this is a considered “high-risk” relapsed disease. Patients with a high risk of MM do poorly with conventional treatment options and are therefore encouraged to participate in clinical trials that employ novel therapeutic strategies. Outside of a clinical trial, VRd remains the preferred choice of initial chemotherapy for patients with high-risk MM who are not candidates for HCT ,29. KRd On the front line, autologous HCT is preferred for those who can tolerate it. Those undergoing HCT typically receive a triplet regimen for 3–4 months prior to stem cell collection in order to reduce tumor cell numbers in the bone marrow. Those who had a durable benefit from HCT in front-line or did not receive HCT in front-line but are now eligible are recommended an HCT in second line following induction ,34. CollThe recently approved monoclonal antibody, daratumumab, which targets CD38 (a receptor highly expressed on myeloma cells), has become a favorite for later line induction regimens ,36. OtheExtramedullary disease can be seen in 5% of MM patients ,38. It cDespite significant advances in the management of myeloma, the treatment for relapsed/refractory multiple myeloma (RRMM) continues to be challenging. Venetoclax, an inhibitor of the anti-apoptotic protein BCL-2, is very effective among patients with t. A review by Basali et al.  has showOther treatment considerations, including bisphosphonates, such as zoledronic acid, or bone-stimulating agents (BSAs), like denosumab, are recommended upon diagnosis in order to prevent or palliate lytic lesions and fractures ,43. MoreSeveral studies are underway to evaluate the effect of adding a fourth drug (such as daratumumab) to a triplet backbone but are not yet standard of care. Chimeric antigen receptor T cell therapy for the MM receptor BCMA is currently being investigated and has shown promise for refractory disease .With the advancement in the treatment options, myeloma is more like a chronic disease and the goal of the therapy is to prevent end-organ damage and to attain disease-free survival . The comThe patients are at a higher risk of infection due to the disease and immunosuppression secondary to chemotherapy. Neurological complications include peripheral neuropathy and cognitive issues secondary to therapy. Cognitive deficits can be noted in patients treated with immunomodulators ,55. TherMultiple myeloma is an aggressive plasma cell dyscrasia that is on the rise in the US and much of the developed world. In the US, MM constitutes over 2% of all cancer deaths (which is double the proportion in the rest of the world). While incidence has grown by over 40% in the US over the past decades, mortality has fallen, and 5 year survival has more than doubled thanks to the introduction of new therapies and transplant techniques. The neoplasm is more common among older adults, men, and African Americans, and has been shown to have a hereditary component to risk. The first step in diagnosis is urine or serum electrophoresis and FLC assays, followed by imaging. Multiple myeloma is distinguished from SM and MGUS by a high (>3 g/dL) level of M-protein  and the presence of CRAB symptoms (end-organ damage). The ISS staging was originally based on B2M and albumin levels, while the revised system includes prognostic factors such as LDH levels and chromosomal abnormalities. Front-line management includes HCT for those who are eligible, induction triplet regimens, and maintenance therapy, along with bisphosphonates or BSAs for prevention of skeletal events. Treatment for the relapsed disease includes monoclonal antibodies like the CD38-targeting daratumumab, proteasome inhibitors, and immunomodulating agents.Objectives and Novelty: This article describes the latest review of multiple myeloma. An extensive literature search was done and up-to-date information regarding epidemiology, staging, and management was described.


Worldwide, the leading cause of chronic liver disease is represented by nonalcoholic fatty liver disease (NAFLD) which has now become a global epidemic of the 21st century, affecting 1 in 4 adults, and which appears to be associated with the steadily increasing rates of metabolic syndrome and its components , and dyslipidemia). NAFLD has been reported to be associated with extrahepatic manifestations such as cardiovascular disease, T2DM, chronic kidney disease, extrahepatic malignancies , endocrine diseases , obstructive sleep apnea, and iron overload. The prevalence of NAFLD is very high, affecting 25–30% of the world population and encloses two steps: (1) nonalcoholic fatty liver (NAFL), which includes steatosis only, and (2) nonalcoholic steatohepatitis (NASH) defined by the presence of steatosis and inflammation with hepatocyte ballooning, with or without fibrosis which can progress to liver fibrosis, hepatocellular carcinoma, and liver transplantation. Current data define a more complex relationship between NAFLD and T2DM than was previously believed, underlining a bidirectional and mutual association between the two entities. This review aims to summarize the current literature regarding the incidence of T2DM among patients with NAFLD and also the prevalence of NAFLD in T2DM patients, highlighting the recent key studies. Clinicians should screen, diagnose, and treat T2DM in patients with NAFLD in order to avoid short- and long-term complications. The incidence rate of chronic liver diseases increased through the years with a worrying rise of liver-related morbidity and mortality rates worldwide . One of Growing evidence clearly shows that NAFLD is a multiorgan disease, supporting a strong link between NAFLD and cardiovascular diseases (CVDs), T2DM, chronic kidney disease (CKD), extrahepatic malignancies , obstructive sleep apnea (OSA), and various endocrinopathies , osteoporosis, psoriasis, hypothyroidism, and iron overload) , 11. AltCurrent data reveal a more complex relationship between NAFLD and T2DM than was previously believed, highlighting a bidirectional and mutual association between the two entities , 15, 16.NAFLD is associated with lipotoxicity which is secondary to the accumulation of triglyceride-derived toxic metabolites in the liver, pancreas, and muscles, which leads to the activation of the inflammation cascade and insulin resistance , 19. TheThe prevalence of T2DM in patients with NAFLD depends on the severity of NAFLD starting from 9.8% in mild NAFLD to 17.8% in moderate to severe NAFLD –22. AlthDespite evidence from several studies which demonstrated that high levels of NAFLD's surrogate markers—gamma-glutamyl transferase and alanine aminotransferase (ALT)—were associated with a high incidence of T2DM, the predictive value of these biologic parameters is limited due to the possibility of normal levels among these patients –29. As fMany large-population-based retrospective studies with a follow-up period of 3 to 6.2 years showed an increased incidence risk of T2DM among patients diagnosed with NAFLD  33–37, , 43. ShiOnce established, T2DM may promote the progression to NASH and become an independent risk factor for chronic liver disease, including cirrhosis and HCC . It has Evidences from several recent cohort studies highlighted an increased incidence of NAFLD among patients diagnosed with T2DM, with an estimated prevalence ranging between 41.6% and 86%  8, 44–4–444–49. The reciprocal relationship between T2DM and NAFLD leads to the progression of LF and is secondary to the development of liver-related complications with high morbidity and mortality rates. In order to avoid systemic multicollateral damage, it seems appropriate to screen patients with NAFLD for T2DM, and vice-versa. According to current European Guidelines, screening of T2DM in patients with NAFLD is mandatory, and it consists of random blood glucose or hemoglobin A1C . FurtherIn light of current evidence, the clinical burden of NAFLD is not limited to liver-related complications, but is in fact, related to its extrahepatic manifestations such as CVD, T2DM, CKD, extrahepatic malignancies, OSA, and various endocrinopathies, with increased mortality rates. Our review highlights that the components of NAFLD (NASH and NAFL) lead to insulin resistance and T2DM through various physiopathological pathways, but also T2DM may promote the progression to NASH as an independent risk factor. Thus, clinicians should be aware of these NAFLD-related extrahepatic manifestations considering that an early acknowledgement of T2DM is the key point in the management of these patients.


Agave sensu lato is one of the most diverse and complex genera of Asparagaceae, with more than 250 species. The morphological, ecological, and evolutionary diversity of the group has complicated its taxonomical study. We conducted phylogenetic analyses of DNA sequence data to reconstruct the phylogenetic relationships of the Agave genus. We included 107 species of the Asparagaceae family from which 83 correspond to the Agave sensu lato clade  and as outgroups the genera Dasylirion, Hesperoyucca, Chlorogalum, Camassia, Hesperaloe, Yucca, Beschorneria, and Furcraea, in order to estimate the age and propose the history of their diversification. Previous studies postulated the relevance of the Miocene in the speciation rates of the agaves, as well as the relevance of the type of inflorescence in its diversification. However, these assertions have not been well supported. The analysis of chloroplast regions resulted in low resolution, which could be the consequence of the few variable sites. On the other hand, the internal transcribed spacer (ITS) implemented in our analysis ensued in higher resolution and better support values. Our phylogenetic analyses recovered five groups; one is the Striatae group, which is the sister group to Agave sensu stricto clade. Within this clade, we found three main groups with high support; these groups are not related with previous morphological proposals. We also analyzed the dates of origin and diversification rates. A Bayesian analysis of macroevolutionary mixtures indicated two significant shifts; the first was identified at 6.18 Ma, where the speciation rate increased to 4.10 species/Mya, this shift occurred during the late Miocene period, characterized by the emergence of arid biomes in North America. The second was identified at a stem age of 2.68 Ma where the speciation rate increased to 6.04 species/Mya. Concerning the ancestral reconstruction state of the inflorescence type in the Agave sensu stricto clade, the spike inflorescence character was predominant in the early-diverging groups, whereas the late-diverging groups present panicle inflorescences as the predominant character and higher speciation rates. The process of evolutionary radiation has been considered as one of the most important sources of biological diversity, through relatively rapid differentiation from a single ancestor into new species that inhabit a variety of environments and may differ in the characters they use to exploit them . An evolThe factors that influence the net species diversification rate–that is to say, the net result of speciation and extinction for each taxon–are multiple and complex . These iOne of the challenges when studying the diversification of a group is understanding the factors influencing speciation and extinction rates, since the possible factors are many and it is difficult to disentangle which one or if several factors are affecting diversification . The comAgave sensu lato clade since its origin in the Miocene 7.9–10.2 Ma ; from this, 74 species correspond to Agave sensu stricto, this sampling includes at least one member of each of the morphological groups proposed by Agave  and as outgroups the genera Dasylirion (3), Hesperoyucca (1), Chlorogalum (2), Camassia (2), Hesperaloe (3), Yucca (5), Beschorneria (4), and Furcraea (4).A total of 107 species of the Asparagaceae family were sampled for the phylogenetic analysis from which 83 correspond to the K , rps16 (835 bp), trnH-psbA (561 bp), and rpl32-trnL (838 bp) from 43 Agave sensu stricto species + 2 Manfreda + 1 Polianthes and Furcraea (2) + Beschorneria (1) and Yucca (1) species as outgroups. For the internal transcribed spacer (ITS) data set, we analyzed 577 bp from 72 Agave sensu stricto species + 4 Manfreda + 4 Polianthes + 1 Prochnyanthes and Dasylirion (3), Hesperoyucca (1), Chlorogalum (2), Camassia (2), Hesperaloe (3), Yucca (5), Beschorneria (4), and Furcraea (4) as outgroups. All newly generated nucleotide sequences for this study were deposited in the NCBI GenBank.The concatenated matrix used for the chloroplast phylogeny included sequences of matK, rps16, trnH-psbA  tree for the concatenated matrix from plastid regions was constructed using RAxML . We condodelTest  under a Dasylirion clade as the outgroup.For the ITS matrix, the best model was selected based on BIC implemented in jModelTest . We ran A Bayesian phylogenetic tree was reconstructed using MrBayes 3.2.2 . The besYucca, with a lognormal distribution and a mean of 14.2 Ma; this includes the age of the strata of the fossil Protoyucca shadishii from the middle Miocene, which is considered as being closely related to the Yucca genus  v2.5.0 software  for R S. Priors r) for both Agave sensu lato clade and other groups identified as having significant rate shifts in speciation. We used the functions getCladeRates to obtain estimates of the speciation rate (λ) and an extinction rate (μ) for a specific clade.Diversification rates were inferred using the function “speciation–extinction” of BAMM, which allows detecting rate shifts (assumed a compound Poisson process in the phylogeny) along tree branches. The evolutionary rate parameters used were: expected number of shift = 1.0, lamdaIntPrior = 1.0, lambdaShiftPrior = 0.05, and muInitPrior = 1.0. BAMM uses rjMCMC to explore the distinct evolutionary models that best explain the whole diversification of the clade. The analysis was conducted by concurrently running two independent chains for 20,000,000 generations and assuming convergence of the chains when the ESS value exceeded 200. For diversification analyses, we retrieved the configuration of rate shifts with the highest posterior probability through the “getBestShiftConfiguration” function of BAMMtools. These configurations were depicted as phylorate plots, which represent the analyzed phylogeny with its branches colored to reflect the instantaneous diversification rate. Rates-through-time plots were generated for speciation (λ), extinction (μ), and diversification , whereas Agave sensu stricto was not monophyletic. ML and BI trees are shown in For the chloroplast phylogeny, a total of 3,670 bp from 46 Hesperaloe/Hesperoyucca + Chlorogalum/Camassia + Yucca groups and a second one formed by the Furcraea/Beschorneria + Agave sensu lato groups. However, the Hesperaloe/Hesperoyucca, Chlorogalum/Camassia, and Yucca groups were not resolved. The ML tree was also unresolved for the Hesperaloe/Hesperoyucca, Chlorogalum/Camassia, and Yucca + Furcraea/Beschorneria + Agave sensu lato clades. Nonetheless, in both analyses, BI and ML trees were in agreement that the Yucca group is independent to the lineage leading to the Furcraea/Beschorneria + Agave sensu lato groups. The Furcraea–Beschorneria clade (0.95 PP/100% BS) came out as a sister group to Agave sensu lato in both analyses (0.99 PP/99.8% BS). However, the Agave sensu stricto group is paraphyletic with respect to Manfreda, Prochnyanthes plus Polianthes, that together constitute a clade with high support value (1 PP/100% BS)  resulted in congruent topologies . BI best100% BS) .Agave sensu lato clade, Agave ellemeetiana (1 PP/98.4 BS) and Agave bracteosa (0.99 PP/98.4 BS) belonging to the Choripetalae group, consistently emerged early in the evolution of the group using either BI or ML methods forming a paraphyletic grade  and was clearly separated from the clade that we will name here as Agave sensu stricto and from Group II containing Manfreda, Polianthes, and Prochnyanthes (1.0 PP/100 BS%) (The clade that conformed by 100 BS%) .Agave sensu stricto clade (1.0 PP/98.4% BS), Agave pelona (1.0 PP/98.4% BS) was positioned as the sister of the Agave sensu stricto clade  comprises Agave angustifolia and a subgroup formed by Agave antillarum, Agave attenuata, Agave chiapensis, Agave colorata, Agave convallis, Agave ghiesbreghtii, Agave guiengola, Agave horrida, Agave kerchovei, Agave marmorata, Agave pendula, Agave titanota, and Agave triangularis.Group IV (0.85 PP/100% BS) included Agave americana, Agave datylio, Agave atrovirens, Agave aurea, Agave capensis, Agave cerulata, Agave cupreata, Agave delamateri, Agave deserti, Agave desmettiana, Agave maximiliana, Agave mckelveyana, Agave phillipsiana, Agave potatorum, Agave scaposa, Agave seemanniana, Agave shawii, Agave sobria, Agave vilmoriniana, Agave wocomahi, Agave zebra, Agave grijalvensis, Agave isthmensis, Agave lurida, Agave rhodacantha, Agave sisalana, and Agave tequilana (Group V (0.99 PP/89.2% BS) was composed of equilana .Agave sensu stricto were not part of the above-mentioned strongly supported clades and were found paraphyletic to these clades, both by BI and ML  for the presence of a spike inflorescence (The reconstruction of the inflorescence types showed ambiguous results in the sense that it was not clear if the common ancestor of the rescence .Agave sensu lato clade (A. bracteosa and A. ellemeetiana), as well as Striatae clade .The reconstructions for the common ancestor of two species with early divergence from the ae clade , indicatAgave sensu stricto clade, A. pelona is the sister taxa to the rest of the group and presents a spike inflorescence, in agreement with 75% of the reconstructions for the common ancestor of the Agave sensu stricto clade having spike inflorescences. The common ancestor for Group III most likely showed a spike inflorescence (98%). In contrast, for Group V, the one with the most recent origin, the common ancestor, and the extant species presented a panicle inflorescence (98%), with the exception of A. vilmoriniana, which reverted to a spike inflorescence (Within the rescence .r) using the ITS data estimated for Agave sensu lato was 1.50 species/Myr, with a speciation rate of λ = 3.66 species/Myr and an extinction rate of μ = 2.16 species/Myr ; this shift is located at the branch of the Agave sensu stricto clade at 4.91 Mya , which is congruent with previous analyses that included plastid markers for the to clade . The phyto group . Indeed, species , which c species .Agave sensu lato clade. In the aligned ITS2 sequence, there is a large deletion shared by Yucca species, first noticed by Agave sensu lato detected in our study are consistent with these previous studies  than the plastid data set and showed higher resolution and support values. The first phylogenetic study from ITS markers for the clade by  studies , 2006. IManfreda–Polianthes–Prochnyanthes, are interesting, given that the Polianthes and Prochnyanthes are paraphyletic with respect to Manfreda. This nesting was also reported in previous studies , and A. stricta  species, in contrast with most A. sensu stricto species, that are usually monocarpic (semelparous). The Striatae group seems to be less frequently pollinated by bats than the other species in the A. sensu stricto group  clade dies see . The Strtrophila , A. gracracielae , A. cremmnophila , A. lexiA. lexii , A. rzedowskiana , A. tenunuifolia , A. albobopilosa , and Agaavandivi , which cto group , 2006. IAgave sensu lato clade are A. ellemeetiana and A. bracteosa, which Agave to a separate genus,” but future formal trait analyses are needed. A. guiengola, because of its virtually tubeless flower and the insertion of the filaments at the base of the flowers, a species that in our analysis is positioned in Group IV of Agave sensu lato. This last placement is not surprising, as Agave guiengola from either of the species mentioned .” In particular, A. ellemeetiana and A. bracteosa are interesting species, since they have margins without any type of teeth, whereas the Striatae group presents serrulate margins; the presence of margins without teeth or serrulate is a character present in many of the early divergent species in the phylogeny of the Agave sensu lato. For instance, A. pelona, which is paraphyletic with respect to the Agave sensu stricto clade, was named this way by The two species at the base of the Agave sensu lato started diversifying at 6.18 Ma according to our crown age estimate. This is congruent with the first significant diversification shift obtained from BAMM, which is at the base of the Agave sensu lato clade. This original shift in the diversification rate was previously reported by Agave sensu lato clade, and it could be the starting point for the diversification of agaves. In Agave sensu lato, the earlier divergent groups  have predominant ancestors with spike inflorescences according to our ancestral character state reconstruction, which are commonly pollinated by bees and hawk moths , where the predominant character is a paniculate inflorescence.wk moths , 2006. TAgave, Manfreda, Beschorneria, Furcraea, Hesperaloe, and Yucca, whereas spike or raceme inflorescences are found in Hesperaloe, Polianthes, Prochnyanthes, and also in Agave , panicle inflorescences in the late-diverging groups (Group V), and frequent reversions between the two forms (Groups III and IV) remain to be studied.Are these reconstructed chains of events consistent with what we know about inflorescence development and evolution? Inflorescence architecture is the consequence of developmental programs that dictate inflorescence meristem activity and determine organ topology, geometry, and phenology by means of the regulatory processes affecting meristem identity, size, and maintenance, as well as axillary meristem initiation and organogenesis . There iin Agave . High plin Agave . TherefoA. pelona and its sister group Agave sensu stricto at 2.68 Ma when we observed an increase in the speciation rate (λ = 6.04 species/Myr). This is concordant with Agave sensu lato distribution and species richness could be related to glaciation and interglacial events during the Pleistocene that caused the expansion and contraction of the species distribution, thus influencing the evolution of agave populations. Three localities of interest during this period are the southern portion of Sierra Sur de Chihuahua, which served as refuge during the last interglacial period, the Sierra Madre del Sur, which is considered as a refuge during the Last Glacial Maximum , and the California Sierra during both periods. This second increase in the diversification rate is in accordance with a second diversification proposed by Agave seems to be older. The second shift could represent a secondary adaptation to different climates that permitted the lineages to diversify, including adaptation to more mesic conditions in Central and West Mexico, giving rise, for instance, to the radiation of the Manfreda–Polianthes–Prochnyanthes herbaceous group and of some agave groups that live in less arid environments in central Mexico.During the Pliocene and Pleistocene, the agaves had the greatest amount of diversification events. This corresponds to the second rate shift within the stem of Yucca family, because few bat species, in particular Leptonycteris yerbabuenae, visit and pollinate many species of agave, whereas each species of Yucca seems to coevolve with a particular Tegeticula moth  is clearly higher than that of related groups, such as Yucca (r = 0.12 species/Myr) and Furcraea–Beschorneria (r = 0.11 species/Myr), values similar to those reported by Agave sensu lato clade. Agave sensu lato has a considerably higher diversification rate than average estimates reported for other flowering plants, which range from 0.078 to 0.09 species/Mya (Agave sensu stricto branch (λ = 5.67 species/Myr), continuing to increase in the three main groups that conform the Agave sensu stricto clade: Group III with a λ = 6.05 species/Myr, Group IV with a λ = 6.15 species/Myr, and Group V with a λ = 6.15 species/Myr. On the basis of the elevated speciation rate values estimated for the Agave sensu lato clades, we can conclude that as a group of recent origin, it is experiencing an intense process of diversification (The diversification rate of cies/Mya . The spefication .All sequences generated for this study were deposited in the NCBI GenBank under accession numbers shown in OJ-B contributed to the laboratory work, data analysis, and drafting of the manuscript. RG-S contributed to the phylogenetic analysis, reconstruction of ancestral state analyses, and design of some figures. SM helped in drafting and correcting sections of the manuscript. AG-M helped in drafting and correcting sections of the manuscript and with the species collection for the analyses. JN-S contributed with ideas for the design of the project, DNA material, and correcting sections of the manuscript. EA-P helped in drafting and correcting sections of the manuscript. LE, as the project leader, designed and coordinated the project and logistics and drafted and corrected the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The young Indian population, which constitutes 65% of the country, is fast adapting to a new lifestyle, which was not known earlier. They are at a high risk of the increasing burden of diabetes and associated complications. The new evolving lifestyle is not only affecting people’s health but also mounting the monetary burden on a developing country such as India.We aimed to collect information regarding the prevalence of risk of diabetes in young adults (<35 years) in the 29 most populous states and union territories (7 zones) of India, using a validated questionnaire.A user-friendly questionnaire-based survey using a mobile application was conducted on all adults in the 29 most populous states/union territories of India, after obtaining ethical clearance for the study. Here, we report the estimation of the prevalence of the risk of diabetes and self-reported diabetes on 58,821 young individuals below the age of 35 years. Risk for diabetes was assessed using a standardized instrument, the Indian diabetes risk score (IDRS), that has 4 factors . Spearman’s correlation coefficient was used to check the correlations.The prevalence of high (IDRS score > 60), moderate (IDRS score 30–50), and low (IDRS < 30) diabetes risk in young adults (<35 years) was 10.2%, 33.1%, and 56.7%, respectively. Those with high-risk scores were highest (14.4%) in the Jammu zone and lowest (4.1%) in the central zone. The prevalence of self-reported diabetes was 1.8% with a small difference between men (1.7%) and women (1.9%), and the highest (8.4%) in those with a parental history of diabetes. The south zone had the highest (2.5%), and the north west zone had the lowest (4.4%) prevalence.Indian youth are at high risk for diabetes, which calls for an urgent action plan through intensive efforts to promote lifestyle behavior modifications during the pandemics of both communicable and noncommunicable diseases. India is a fast developing economy with a considerable number of diabetes patients. Its health care cost is rising with a deterioration in health standards among the economic productive young population –4. It isDiabetes has become a global pandemic and threat for world health due to demographic variations and cultural differences of societies supplemented by aging phenomena. It is a costly disease that has been identified as the prime causative factor for blindness, lipoprotein abnormalities, or mitochondrial dysfunction causing cardiovascular diseases, renal failure, and amputation in several countries –10. The Genetic and environmental factors, such as heredity, change in lifestyle, age, smoking habits, increased alcohol consumption, screen time, parental conflicts, improper sleep, education, and stress, predispose young adults to diabetes, which is exacerbated with diabetic comorbid conditions . ObesityThe lack of physical activity among the younger population is a matter of concern as 84% of girls and 78% of males in Australia did not meet the criteria for minimum physical activity corresponding to their age. As a consequence, females were found to be more overweight than males . The risOur study was aimed to estimate the prevalence of low, moderate, and high risk of diabetes in young adults. We conducted a nationwide study by collecting information regarding prevalence of risk of diabetes in young adults using a validated questionnaire. Moreover, the contribution of other sociodemographic factors, such as age, physical activities, yoga, family history, vitals, diet, gender, marriage, education, occupation, and socioeconomic status, were further collected to examine diabetic progression.The study was conducted after ethical clearance from the ethical committee of the Indian yoga association with reference number RES/IEC-IYA/001. The data used in this analysis has been collected during phase 1 of the NMB 2017 trial, a large translational, multicenter, cluster-sampled research trial aimed to assess the efficacy of yoga-based lifestyle modification as a primary prevention strategy for diabetes in a community setting. The methodological details of the study have been reported previously . In briehttp://www.sample-size.net), the required sample size for a two-group design with α = 0.05 and (1− α) = 0.80 was estimated to be 1949 for each group . Factoring an attrition of 20%, the final sample size was estimated to be 4678 individuals with prediabetes. To obtain 4678 individuals with prediabetes, it was calculated that there was a need to screen 77,967 adults above the age of 20 years  in prediabetes individuals reported in the Community Lifestyle Improvement Program study . We usedeen 6.0% . Thus, tWe acquired information on diabetes and risk scores by a door-to-door survey using a mobile application with detailed person-level information about age, gender, income details, educational qualifications, and marital status.The Indian Diabetes Risk Score (IDRS) developed by Mohan et al. in 2005 was used for risk analysis . IDRS isNiyantarit Maduhmeha Bharat (NMB) 2017 was a pan-India randomized multicluster translational trial with dual objectives, namely, a survey for prevalence and lifestyle intervention for the population at high risk and known diabetes (Field personnel  were trained in a 5-day residential program to ask appropriate questions in local languages that included practical tests by visiting nearby villages and urban wards.Chi-square and Fisher exact tests were used for mean differences. Binary logistic regression analysis was done to find the association between independent predictors of diabetes. Self-reported diabetes was considered as a dependent variable. Gender, area, marital status, parental history, IDRS, physical activity, and waist circumference were covariates by keeping the reference factors rural for area, female for gender, vegetarian for diet  version. The estimation of prevalence was calculated using the distribution of frequency and percentage using cross tabs descriptive. p < 0.001) , the young diabetes population was screened across the nation on the basis of IDRS and self-reported diabetes, using validated IDRS; 60,194 individuals were selected on the basis of IDRS score, and 58,821 were selected on the basis of self-reported diabetes as young adults (<35 years). Gender-related risk of diabetes was found to be similar in men and women. The prevalence of self-reported diabetes in young females was 1.9% and in men 1.7%. On the basis of IDRS risk, no significant difference was found in the female and male diabetes population Table 1.P < 0.001). Frequency distribution based on the parental history of diabetes has also reflected significantly higher numbers in high IDRS scores as compared to low-risk IDRS. Results suggest the inheritance pattern of diabetic condition, which may be triggered with familial lifestyle or genetic susceptibility of parents and trait transmission in siblings.Interestingly, it has been found that 1.3%, 5%, and 8.4% of diabetic subjects were self-reported with no parental history of diabetes, one diabetes parent, and both diabetes parents with diabetic history, respectively (p< 0.001). The IDRS scores (based on waist circumference) were also significantly higher in high-risk participants based on waist circumference of individuals, i.e., almost 33.6% more than moderate (11.7%) and normal (0.4%) individuals  and obese (>30) (3.3%) young population was at significantly higher risk of diabetes than the normal (1.3%) and underweight (1.1%) young population Table 1.The frequency distribution based on physical activities was also in concordance with the BMI and waist circumference of the participants. It was found that the proportion of individuals who performed no, mild, moderate, or vigorous physical exercise were comparable.p = 0.221, The zone-wise prevalence of diabetes (self-reported) was significantly different (<0.001) and reported as follows in descending order: south, north, east, northeast, central, west, and Jammu. However, no gender-wise significant differences were found Table 2.p < 0.001) of diabetes as compared to married individuals. The comparison was made for relative risk of diabetes  were found to have higher odds of developing diabetes as compared to low-risk young adults. Unmarried young adults had 1.290 higher odds . There is a need to develop a cost-effective and preventive management program to reduce or prevent diabetes complications in young adults. As subjects Table 4.Studies show that yoga helps in the activation of the hypothalamic pituitary axis and sympatho-adrenal component known to inhibit glucose uptake by inhibiting insulin release, inducing insulin resistance and increasing hepatic glucose production . VigorouInterestingly, young diabetes patients are amenable to reversal by intensive lifestyle intervention as seen in this young diabetes study . The diaThe study used cluster sampling, which might have contributed to the sample selection bias. As a result, some subjects with diabetes might have refused to admit to having diabetes. It is also possible that a few subjects are wrongly believed to have diabetes, and there is no validation of such self-reported diabetes. Furthermore, undiagnosed diabetes could be another confounder. Subjects frequently ignore the subtle signs and symptoms of asymptomatic diabetes. The possibility of underestimation of the prevalence of diabetes in the proposed population may be the main limitation.All datasets generated for this study are included in the article/Ethical permission obtained from Institutional Ethics Committee (IEC) meeting held at Indian Yoga Association, Morarji Desai National Institute of Yoga with reference no. RES/IEC-IYA/001 dated 16th Dec 2016.RN is a grant PI involved in conceptualization, editing of manuscript. PB was involved in original writing and data analysis. VS edited the manuscript. AA was involved in conceptualization of manuscript. VS edited the manuscript. SP was involved in data curation and analysis. GS and AS were involved in the acquisition of data. VP was involved in writing, editing and collection of data on site as physician. HRN was involved in conceptualization of manuscript, obtained resources and mentoring of work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Abdominal pain (AP) is a common reason for presentation to an emergency department (ED). With this prospective, observational all-comer study, we aimed to answer three questions: Which diagnoses are most often missed? What is the incidence of extra-abdominal causes? What is the prognosis of abdominal pain in a tertiary urban European ED? Participants were systematically interviewed for the presence of 35 predefined symptoms. For all patients with abdominal pain, the index visit diagnoses were recorded. Related representation was defined as any representation, investigation, or surgery related to the index visit (open time frame). If a diagnosis changed between index visit and representation, it was classified as missed diagnosis. Among 3960 screened presentations, 480 (12.1%) were due to AP. Among 63 (13.1%) related representations, the most prevalent causes were cholelithiasis, gastroenteritis, and urinary retention. A missed diagnosis was attributed to 27 (5.6%) presentations. Extra-abdominal causes were identified in 162 (43%) presentations. Thirty-day mortality was comparable to that of all other ED patients (2.2% vs. 2.1%). Patients with abdominal pain had a low risk of representation, and the majority of representations due to missed diagnoses were of benign origin. The high incidence of extra-abdominal causes is noteworthy, as this may induce change to differential diagnosis of abdominal pain. Abdominal pain (AP) is among the most common reasons to present to an emergency department (ED) ,2,3. In It has been described that abdominal pain is amongst the most frequent problems associated with malpractice claims at the emergency department . Other iTherefore, three questions can be considered unanswered or controversial. First: Which diagnoses are most often missed and lead to representation? Second: What is the prevalence of extra-abdominal causes of abdominal pain? Third, what is the short- and long-term prognosis of abdominal pain in a tertiary urban European ED, stratified according to age?We performed a prospective, observational study with a one-year follow-up, designed as an all-comer analysis in which all patients were interviewed at presentation to the ED by a study team. To our knowledge, there are no data on the true prevalence of abdominal pain in the ED, as the cited studies have all relied on the determination of a chief complaint by triage nurses or emergency physicians. As there is evidence that “off-hour” patients differ in acuity  and thatWe hypothesized that missed diagnoses leading to representation are considerable (>10%), that extra-abdominal causes of abdominal pain are frequent (>25%), and that prognosis is highly dependent on age in this notoriously difficult patient population.www.eknz.ch) approved the conduct of the study .This prospective monocentric all-comer study was conducted in the emergency department of the University Hospital of Basel, a tertiary care center in Northwestern Switzerland with an annual ED census of more than 50,000 patients and a capacity of about 700 beds. The investigation took place within two time periods lasting three weeks each. The first one started on 21 October and ended on 11 November 2013, the second one was realized between 1 and 23 February 2015. Two different time points were chosen because of logistic and financial reasons and to minimize seasonal effects by choosing two different seasons. The local ethics committee  was used to triage all patients, level 1 being the highest acuity and urgency, and level 5 the lowest .A dedicated study team, comprised of medical students, was trained to collect data using a standardized questionnaire. They worked in three shifts to include patients 24 h a day, 7 days per week. When presenting to the ED, patients were registered, and an electronic health record (EHR) was generated. They were triaged by a nurse or a physician according to the German version of the ESI . ParticiAll patients’ charts were abstracted by two independent chart reviewers. Medical record review was performed with the EHR database . A case report form, created with Microsoft Access 2016, was used, in order to document the results. To classify diagnoses, a three-digit ICD-10  code was generated by the two independent chart reviewers. The patients’ underlying conditions were taken from the discharge report. In case of disagreement between the two chart reviewers, an experienced emergency physician served as a referee. The main diagnoses at the index visit were recorded in the database after agreement of all reviewers.In the event of a representation, the diagnoses at representation were coded independently of the index visit in a second step, in order to minimize anchoring bias. In a third step, the diagnoses were compared between index visit and representation. Related representation was defined as any representation, investigation, or surgery in relation to the index visit . There was no time frame limiting a related representation, and chart abstraction to assess follow-up was performed over one year after the index visit. Two reviewers independently decided if the representation was related to the index visit. If there was a change of diagnosis between index visit and related representation, the case was classified as missed diagnosis. In case of discordant classifications of the reviewers, the referee was called as described above. According to our expert group a “low rate” was defined as less than 10% missed diagnoses. A “considerable rate” was defined as over 10% missed diagnoses associated with representations. These definitions are expert opinion only, and hypotheses were made by the study group after broad assessment of the literature in this field.We defined abdominal diagnoses as diseases originating in the peritoneal, retro-peritoneal , and pelvic compartments, including vascular problems. This includes diseases of the digestive system, coded in chapter K in the ICD-10-system, and other diseases of abdominal origin, such as malignancies (chapter C), diseases of abdominal vessels (chapter I), or infectious diseases (chapter A/B) of abdominal origin. Further, diseases originating in the retro-peritoneal (pancreas) or pelvic compartments (reproductive system) were also subsumed to abdominal diagnoses. Viral, infectious, and unspecified gastroenteritis were condensed , as well as urinary tract infection and cystitis . We defined extra-abdominal diagnoses as diseases causing abdominal pain not originating in the abdominal or pelvic compartment. This included diagnoses of all other compartments or organ systems, such as chest , urogenital , cerebral , blood , as well as psychiatric diagnoses , intoxications, or systemic diseases  . After aInformation about length of stay (LOS), as well as hospitalization outcomes, intensive care unit (ICU) transfer, and mortality was extracted from the EHR. One year after presentation, a follow-up regarding survival was conducted. Data were extracted from the EHR, from official residents’ and insurance registries, as well as directly from patients, proxies, and family physicians.Hospitalization, according to the Swiss law, was defined as at least one overnight stay in a hospital bed. LOS was defined as the number of days spent in hospital during the index hospitalization. ICU transfer was defined as any admission to medical, surgical, or neurosurgical ICUs or to a stroke unit during the index hospitalization. In-hospital mortality was defined as the percentage of patients who died after presenting to the ED without being discharged between admission and death. Thirty-day and one-year mortality were defined as the percentage of deceased patients at 30 days and 1 year after presenting to the ED. If patients presented more than once during the inclusion periods, we did not exclude them and treated them as independent presentations for all analyses, except for mortality. Only the first presentation was taken for mortality calculations.The rate of missed diagnoses, the incidence of extra-abdominal conditions, and the prognosis, as assessed by hospitalization, ICU transfer, and mortality, were determined.p-value <0.05 was considered significant. All calculations were performed using the statistical software R .Descriptive statistics are presented as counts and frequencies for categorical data and medians  for metric variables. Overall p-values correspond to the Mann–Whitney U test for median and to the chi-squared or exact Fisher test when the expected frequencies were less than 5. A During the study period, 5634 presentations were registered. In total, 4703 cases were eligible for screening, and 3960 (100%) were included for further analyses. Among these, 480 (12.1%) presented with AP, and 3480 (87.9%) presented with other symptoms. We registered 310 outpatient presentations and 170 inpatient presentations out of 480 AP presentations. Within the group of 310 presentations, 250 represented to our hospital’s ED or outpatient clinics, 60 did not represent in the open time period after the index visit. Among 250 representations, 63 were related to the index presentation, and 187 presentations were caused by other health problems; 27 “missed diagnoses” were found among the 63 related representations. AP was the seventh most prevalent symptom in this all-comer population see .p < 0.001)  years, and the median age of patients lacking AP was 51  years. A statistically significant higher number of patients (53%) were female among the AP patients, as compared to 48% female patients in the non-AP group. AP patients were attributed to a statistically significant higher triage level , followed by nonspecific abdominal pain  and cholelithiasis . In patients <65 years old, gastroenteritis  and NSAP  headed the list, followed by urolithiasis . Patients ≥65 years old were most likely to suffer from diverticulitis , cholelithiasis , and urinary retention   of the 310 outpatients represented. Among these, 63 cases (25%) were classified as related representations. Patients with related representations presented earlier ) than patients presenting with an unrelated problem ). Almost 90% of all related representations took place within 50 days after the index visit see .n = 8, 13%) followed by gastroenteritis  and urinary retention . A missed diagnosis was attributed to 27 (5.6%) cases. Eight (1.7%) of all AP patients underwent surgery after missed diagnoses  extra-abdominal diagnoses were made, the most frequent ones being of urogenital and pulmonary origin. Nineteen (5%) AP presentations were due to pulmonary problems in younger patients, and 20 (11.2%) in patients 65 years old and older. Psychiatric diagnoses (F-codes according to ICD-10) were made in 17 (4.5%) cases in younger patients, as compared to 3 (1.7%) cases in older patients. Three different pulmonary entities were among the top 20 underlying conditions. Six (1.6%) AP presentations were due to underlying cancer in younger patients, as compared to nine (5%) in older patients. Of all 15 presentations due to cancer, 4 were due to newly diagnosed cancer, and 11 were due to relapse, deterioration, or complications of previously known cancer. Twenty diagnoses were responsible for 63% of all diagnoses see .Of all AP presentations, 170 (35.4%) patients were hospitalized, and 310 (64.6%) patients received outpatient treatment. Hospitalization in patients ≥ 65 years of age was comparable between patients with AP and without AP ). AP patients < 65 years of age were significantly more often hospitalized compared to non-AP patients (26% versus 17%).Median LOS among all patients presenting with AP was 5  days, also comparable to that of patients not presenting with AP. Older patients had a non-significant higher risk of ICU transfer: 10 (7.1%) versus 122 (10.5%) in the AP and non-AP subset, respectively.In-hospital mortality was low for patients < 65 years of age; one (0.3%) patient with AP and 6 (0.3%) patients without AP died. In-hospital mortality was higher in older patients: 5 (3.6%) patients with AP and 43 (3.7%) patients without AP died.One-year mortality among patients ≥ 65 years of age was 21 (15.3%) for patients with AP and 170 (15%) for patients without AP. Differences between the AP and non-AP subgroups regarding LOS, ICU transfer, and mortality were not statistically significant  of related representations, the low rate (5.6%) of missed diagnoses, the very low rate of missed diagnoses with subsequent surgery (1.7%), the high incidence (43%) of extra-abdominal causes, and the large differences in the prognosis and distribution of disease between younger and older patients.Overall, 27 (5.6%) presentations received missed diagnoses. However, we had no evidence for missed diagnoses in hospitalized patients. Therefore, discharged patients were affected in 8.7%. Diagnoses with an over 50% likelihood of being missed at the index visit were malignancies, food intolerance, and gastroenteritis. Every second malignancy was diagnosed at the index visit in the ED, while the other half was made after referral to an outpatient clinic and was therefore counted as representation. The rate of missed diagnoses was low in our cohort, in spite of an overall high rate of representation and an excellent follow-up of over 90%, as compared to other cohorts examining unscheduled returns to the ED . HoweverAs prospective cohorts with abdominal pain in the ED tend to suffer from inclusion bias, because of a tendency to both focus on “acute abdomen” ,26 and aThe prognostic value of the presence of abdominal pain in ED patients was not shown in our all-comer cohort: survival, intensive care, and use of resources were comparable to those of a general ED population. While survival was excellent in patients under 65 years of age, it was lower in patients 65 and older. Abdominal pain as a group does not seem to be a high-risk symptom, unlike dyspnea, altered mentation, and nonspecific complaints, such as generalized weakness [Another controversial issue is the question of “difficult diagnosis”. While the diagnosis of cholelithiasis used to be difficult before ultrasound became widely available, vascular catastrophes were often missed before CT scans were available. According to our data, urogenital problems seem to be the most likely cause of a related representation.In comparison, the incidence of AP was comparable to that found in European  and US sIn older patients, diverticular disease was the most prevalent. A high prevalence of this disease has previously been reported , but in Taken together, all categories of diagnosis may be missed and may lead to representations. However, the rate of representation leading to a modification of diagnosis and surgery was low. Further, the incidence of extra-abdominal causes in abdominal pain was higher than expected. While CHD may be less prevalent than expected, (pulmonary) infections ranging from influenza and COPD to sepsis are to be considered, according to our results. Short- and long-term prognosis of patients with abdominal pain is favorable and largely depends on the patients’ age. In the older population, differential diagnosis may even be broader; in this respect, urinary retention should be identified early on, as its prognosis is dreary .This study shows that awareness of the most common extra-abdominal conditions causing AP  may facilitate a fast and accurate diagnosis and therapy. To improve the clinical practice, a consistent routine during the diagnostic process should be implemented in order to cover a wide range of differential diagnoses.In our setting, AP is common at the ED and lead to a wide range of diagnoses. Missed diagnoses are uncommon, but extra-abdominal causes need to be considered. Therefore, protocol-based work-up needs to cover a considerable number and range of extra-abdominal conditions.Limitations: Several limitations of this study need to be discussed. First, this was a single-center study, and external validity is therefore limited. However, our ED cohort seems to represent the population of other urban, European EDs, with over two-thirds of patients having their origin in central or northern Europe [n Europe  and the n Europe . An inclWe focused on a limited number of predefined symptoms that were actively asked for by medical students, and certain presentations may have been missed. The highly standardized assessment of 35 predefined symptoms may have influenced the results, as the typically applied “physician filter” did not come into play.


Our goal was to develop a prognostic nomogram to predict overall survival (OS) and cancer-specific survival (CSS) in patients with gastric cardia cancer (GCC). Patients diagnosed with GCC from 2004 to 2015 were screened from the surveillance, epidemiology, and end results (SEER) database. A nomogram was developed based on the variables associated with OS and CSS using multivariate Cox analysis regression models, which predicted 3- and 5-year OS and CSS. The predictive performance of the nomogram was evaluated using the consistency index (C-index), calibration curve and decision curve analysis (DCA), and the nomogram was calibrated for 3- and 5-year OS and CSS. A total of 7,332 GCC patients were identified and randomized into a training cohort  and a validation cohort . Multivariate Cox regression analysis showed that marital status, race, SEER stage, grade, T stage, N stage, M stage, tumor size, and surgery were independent risk factors for OS and CSS in GCC patients. Based on the multivariate Cox regression results, we constructed prognostic nomograms of OS and CSS. In the training cohort, the C-index for the OS nomogram was 0.714 (95% CI = 0.705–0.723), and the C-index for the CSS nomogram was 0.759 (95% CI = 0.746–0.772). In the validation cohort, the C-index for the OS nomogram was 0.734 (95% CI = 0.721–0.747), while the C-index for the CSS nomogram was 0.780 (95% CI = 0.759–0.801). Our nomogram has better prediction than the nomogram based on TNM stage. In addition, in the training and external validation cohorts, the calibration curves of the nomogram showed good consistency between the predicted and actual 3- and 5-year OS and CSS rates. The nomogram can effectively predict OS and CSS in GCC patients, which may help clinicians personalize prognostic assessments and clinical decisions. It was reported that there were approximately 27,510 new cases of gastric cancer resulting in 11,140 deaths in the United States in 20191. Anatomically, gastric cancer can be divided into gastric cardiac carcinoma (GCC) and non-cardia gastric cancer (NGCC). In recent decades, although the overall incidence of gastric cancer has declined worldwide2, the incidence of GCC has increased3. This may be due to the increased incidence of gastroesophageal reflux disease and obesity4. According to previous reports, there are significant differences in incidence and prognostic specificity between GCC and NGCC, indicating that they are different tumor entities7. GCC has a poor prognosis and is a serious threat to human health8.Gastric cancer is the 6th most common cancer and the 3rd leading cause of tumor-related death worldwide9. The TNM classification proposed by the American Joint Commission on Cancer (AJCC) is the most widely used staging system, and it is mainly based on tumor invasion (T), regional lymph node (N) and distant metastasis (M) to predict the survival of cancer patients11. However, the evaluation of cancer prognosis based on TNM stage alone has limitations and cannot fully evaluate clinicopathological factors, such as age, sex, race and other factors.At present, the prognosis of GCC is mainly predicted by the TNM staging system13. In recent years, nomograms have been widely used to predict the survival rate of various cancers15. The purpose of this study is to develop an effective prognostic nomogram to predict the overall survival (OS) and cancer-specific survival (CSS) of patients with GCC to help clinicians provide personalized treatment recommendations.The nomogram is a statistics-based tool that calculates the probability of clinical events by considering the preweight value of each factorA total of 7,332 patients were included in the study, and they were randomly assigned to two different cohorts: the training cohort n = 5,132) and the validation cohort . A flow chart showing the process of including patients in the study is depicted in Fig. 32 and thTo identify the prognostic factors, we performed univariate and multivariate Cox regression analyses in the training cohort. According to the univariate Cox analysis, age at diagnosis, marital status, race, histological type, SEER stage, grade, T stage, N stage, M stage, tumor size, and surgery were significantly associated with OS, while sex, age, marital status, race, histological type, SEER stage, grade, T stage, N stage, M stage, tumor size, and surgery were closely related to CSS. These significant variables were further entered into the multivariate Cox analysis. Multivariate Cox analysis showed that age, marital status, race, SEER stage, grade, T stage, N stage, M stage, tumor size and surgery were independent prognostic factors for OS Table . RegardiWe developed two nomograms for OS and CSS: one was based on the results of multivariate Cox analysis Fig. , and theAnalysis of the time-dependent ROC curves for OS and CSS showed that the AUCs of the nomograms  were significantly larger than those of TNM stage  in the training cohort , the clinical net benefit of the nomograms was greater than that of the TNM stage Fig. C,D. The 0%, the c17. After the onset of the disease, patients often have clinical symptoms such as upper gastrointestinal bleeding, dysphagia, and stomach discomfort. The prognosis of patients with GCC is poor18, so it is very important to develop an effective system to predict the prognosis of these patients.GCC is a kind of malignant tumor at the junction of the stomach and esophagus that mostly occurs in middle-aged people over 40 years old and elderly people and that accounts for approximately 10% of all digestive system tumorsIn this study, we first developed prognostic nomograms of OS and CSS in patients with GCC. We used the SEER database to conduct Cox regression analysis on many GCC patients to identify independent risk factors for OS and CSS. We constructed two prognostic nomograms: one based on multivariate Cox analysis and the other based on TNM stage. Through the examination of the C-index, ROC curve, DCA curve and CIC, it was found that our nomogram had better prognostic ability than that of the TNM stage. In addition, we verified and calibrated the nomogram and evaluated the accuracy of OS and CSS nomograms for 3 and 5 years. The results show that the predicted results of the nomogram are in good agreement with the actual observed results and are supported by the calibration curve, ROC curve analysis and C-index value. The C-index of the nomograms is more than 0.7, indicating that it has sufficient discrimination ability. The DCA results show that the nomogram we developed has good clinical practical value.22. Kim et al.19 developed and validated a nomogram that predicted the risk of lymph node metastasis in patients with early gastric cancer and could be used to avoid unnecessary gastrectomy after endoscopic dissection. By analyzing 9,026 patients with metastatic esophageal cancer between 2010 and 2015, Zhu et al.20 found that the nomogram was better at predicting distant metastasis of esophageal cancer than traditional TNM staging. Similarly, the nomogram developed by Xue et al.21 that includes nutritional and immune parameters can effectively predict the overall and postoperative survival rate and relapse-free survival of gastric cancer patients after radical gastrectomy, and its prediction accuracy and discrimination ability are better than those of TNM staging.Recently, some nomograms containing various input variables have been developed to predict the prognosis of different digestive tract tumors22 developed a nomogram for predicting the total survival rate of GCC based on radiology and clinical predictors. However, the nomogram for predicting the OS of GCC based on radiology and clinical predictors is not available, and the nomogram for CSS is applicable for a limited population, since it is only suitable for patients undergoing preoperative radiotherapy.In the current reports on GCC, it is remarkable that Liu et al.23 found that TNM stage was associated with the prognosis of high gastric cancer, and T stage was an independent factor for lymph node metastasis. Zhu et al.24 found that TNM staging was associated with patients with adenocarcinoma of the esophagogastric junction, but only N staging was an independent risk factor for prognosis. TNM staging is a common method to predict the prognosis of GCC patients, but TNM staging has limitations and cannot provide clinicians with personalized prognosis prediction. In this study, we successfully constructed a practical nomogram based on thirteen factors: sex, age, marital status, race, histological type, SEER stage, grade, T stage, N stage, M stage, tumor size, surgery, and chemotherapy, and its prediction power was better than that of traditional TNM staging.TNM staging was determined according to the results of laboratory examination and postoperative pathological examination. For example, Gong et al.Our research still has some limitations. First, the lack of treatment information  in the SEER database may alter our results. Second, our study is a retrospective study with inevitable selection bias. Third, due to the lack of external verification, we are concerned about the generality of our model and may need further research to prove our results.The present study demonstrated that the nomogram is a better prognostic determinant than TNM staging systems in GCC patients. The nomogram we developed accurately and reliably predicted the 3- and 5-year OS and CSS of GCC. This model could enable clinicians to more precisely estimate the survival of GCC patients.25. The National Cancer Institute's SEER*Stat software version 8.3.6 (https://seer.cancer.gov/seerstat/) , Nov 2018 Sub (2004–2016 varying) database) was used in this study.The SEER database is an open public database and provides cancer data  from the population based registries of 18 sites that cover approximately 28% of the USA populationThe International Classification of Diseases for Oncology (ICD-O) site code C16.0 was used to identify patients diagnosed with GCC between 2004 and 2015. In total, 21,860 patients were enrolled in this study according to the specified inclusion and exclusion criteria. The exclusion criteria in our study were as follows: more than one primary tumor ; unknown survival time (n = 32); unknown T stage and T0 ; unknown N stage (n = 569); unknown M stage (n = 92); unknown tumor size ; unknown surgery status (n = 6); and unknown marital status (n = 249). In total, 7,332 GCC patients were included for this analysis.Data regarding sex, age, marital status, race, histological type, SEER stage, grade, T stage, N stage, M stage, tumor size, surgery, chemotherapy, vital status, and survival time were extracted from the SEER database (2004–2015) for further analysis. OS duration was defined as the time from diagnosis until death or last follow-up. CSS duration was defined as the time from diagnosis until death because of GCC or last follow-up.P < 0.05 were further analyzed in the multivariate Cox proportional hazard model. Based on multivariate Cox analysis, a nomogram was developed to predict the 3- and 5-year OS and CSS rates. In contrast, we built another nomogram based on TNM stage. Then, we used MedCalc software (version 15.2.0) to generate the receiver operating characteristic (ROC) curve for the two nomograms and determined the area under the curve (AUC). The performance of the nomogram was assessed by the C-index and the calibration curve . The C-index has a range from 0.5 to 1.0, with 0.5 indicating random chance and 1.0 considered perfect discrimination. Decision curve analysis (DCA) and a clinical impact curve (CIC) were employed to evaluate the net benefit of the nomogram in a clinical context.Cases were randomly divided into training and validation cohorts (ratio 7:3). Univariate and multivariate Cox regression models were applied to calculate the hazard ratio (HR) and 95% confidence interval (CI) to assess the independent contributions of each factor to OS and CSS. In the univariate Cox proportional hazard model, variables with https://www.r-project.org/). All tests were two-sided. A P value ≤ 0.05 (two-sided) was considered statistically significant.The above statistical analyses were conducted using SPSS software version 24.0  and the statistical software package R version 3.5.3 (This article does not contain any studies with human participants or animals performed by any of the authors.Supplementary information.


PRNP locus in goat populations/breeds, so contributing to the design of genetic control measure both in scrapie outbreaks and in disease prevention.Scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats. Polymorphisms in single nucleotides in the coding sequence of the prion protein gene play a major role in relative susceptibility or resistance to classical scrapie. The most recent modification of European Union Regulation 999/2001 (Regulation EU 772/2020) deals with the possible use of genetics in goats for scrapie outbreak management. In this work, sequence analysis of the prion protein gene was performed, with the main aim to obtain information about the genetic variability of eight Italian local populations/breeds and establishing phylogenetic relationships between the breeds/populations. This research could help to promote the adoption of selective breeding programs as a possible future strategy in scrapie control and outbreak prevention. In particular, genomic DNA was extracted from 219 goat whole blood samples and 13 polymorphic sites were observed, giving 24 different alleles. We observed the K222 allele, associated to scrapie resistance, with a mean frequency of 10%. Interestingly, we also described in Italy the circulation of the S146 allele: In particular, S146 only segregated in the Alpine breed at a frequency of 3.7%. This study adds information on genetic variability of the PRNP) can influence animals’ susceptibility to scrapie. Since the polymorphic codons in sheep are well known, a genetic selection plan has been implemented in Europe, in order to reduce the prevalence of susceptible genotypes to scrapie. In Italy, no breeding plan for scrapie resistance in goats has been adopted, yet. Likewise, according to the most recent modification of Regulation EU 999/2001 (Regulation EU 772/2020) of the European Commission (EU), based on all the available experimental and in field data, K222, D146 and S146 polymorphisms could be used as scrapie resistance alleles in genetic management both in scrapie outbreaks and in disease prevention. In order to collect data on the variability of PRNP, the present study aimed to analyze the sequence of the PRNP gene in eight Italian local goat populations/breeds reared in central and southern Italy , some of which were investigated for the first time; moreover, two cosmopolitan breeds (Alpine and Saanen) were included. Blood samples were collected from 219 goats. Genomic DNA was extracted from whole blood. DNA was used as template in PCR amplification of the entire PRNP open reading frame (ORF). Purified amplicons have been sequenced and aligned to Capra hircus PRNP. Particularly, the alleles carrying the resistance-related 222 K polymorphism occurred in all populations with a frequency between 2.5% and 12.5%. An additional resistance allele carrying the S146 variant was observed with a frequency of 3.7% only in the Alpine breed. For three of the estimated alleles, we could not establish if the found double polymorphisms in heterozygosis were in phase, due to technical limitations. In this context, in addition to selective culling in scrapie outbreaks according to the European regulation in force, in the future, selection plans could be adopted to deal with scrapie and to control its diffusion, meanwhile paying attention to preserve a high variability of PRNP.In goats, as in sheep, genotypes of the prion protein gene ( Scrapie in sheep and goats belongs to a group of diseases known as transmissible spongiform encephalopathies or prion diseases. They represent a family of transmissible slowly progressive and invariably fatal neurodegenerative diseases affecting humans and animals . To datePRNP  give rise to five main different alleles, namely VRQ, ARQ, AHQ, ARH, and ARR (defined with the IUPAC amino acid codes). The VRQ/VRQ, VRQ/ARQ, and ARQ/ARQ genotypes are associated with high susceptibility to classical scrapie [In sheep, three polymorphic codons of the  scrapie , while t scrapie , a selecThe breeding plans for sheep led to a frequency increase of the resistance allele. In particular, among the 13 countries characterized by a consistent number of cases, the trend analysis shows a statistically significant decrease of classical scrapie even if with certainty only for six of them .PRNP in goats could represent an opportunity to classify caprine genotypes in those susceptible or resistant to scrapie [PRNP gene can determine the susceptibility or resistance to classical scrapie in goats, but only a few of them have been proven to be associated with resistance [PRNP polymorphisms have been described in goat breeds [PRNP variant that contains three repeats of an octapeptide instead of the usual five repeats was found by Goldman [As already done in sheep, single nucleotide polymorphisms (SNPs) of  scrapie ,16. Furt scrapie , genotypsistance . In partt breeds ,20, but t breeds ,21. Furt Goldman . Amino a Goldman , I142M , Capestrina (CP), Facciuta della Valnerina (FV), Fulva del Lazio (FL), Garganica (GA), Grigia Ciociara (GC), Grigia Molisana (GM), and Teramana (TE). Some of them  are listed in the FAO global data bank for farm animal genetic resources and are classified as endangered [PRNP genotyping as an alternative to stamping out and as a valid approach to control scrapie disease in the presence of outbreaks. Indeed, in Italy, as in the other European countries, no selection plans for scrapie resistance in goats have been developed until now and the disease is still dealt with by the culling of animals in the herd affected by the outbreak. Therefore, data about the distribution of the allele variants of PRNP in goats obtained from this investigation could contribute to direct breeding programs to reduce the possible risk of scrapie.On this basis, the aim of our research was to examine the dangered ,45  following the manufacturer’s instructions.PRNP gene (771 bp). Amplification was performed in a 50-µL reaction volume containing 1.5 mM MgCl2, 200 µM dNTPs , 0.8 µM of forward and reverse primers p8 (5′-CAGGTTAACGATGGTGAAAAGCCACATAGG-3′) and p9 (5′-GGAATTCTATCCTACTATGAGAAAAATGAGG-3′)  [® PCR Purification Kit , according to the manufacturer’s instructions.In total, 100 ng of extracted DNA were used as a template in PCR amplification of the entire open reading frame of the ham, MA) , and 1 UThe quantity and quality of the PCR products were determined photometrically using a Biophotometer (Eppendorf).® 3500 Genetic Analyzer (Thermo Fisher Scientific). Sequences were aligned to the Capra hircus gene for PRNP (Accession number NM_001314247.1) with ClustalW tool of BioEdit 7.0.9 software [Purified amplicons were sequenced in both directions using about 80 ng of DNA. Sequencing reactions were performed with 3.2 pmol of primer p8 and p9 using a Big Dye Terminator Cycle Sequencing Kit v3.1 (Thermo Fisher Scientific) and detected with an ABI PRISMp ≤ 0.05):Genotypic and allelic frequencies regarding codons 37, 110, 127, 137, 142, 146, 154, 168, 194, 211, 215, 222, and 240 were calculated within and across breed, based on counting the respective genotypes of individual animals. The Hardy–Weinberg equilibrium state was examined at each codon and breed using a chi-square test  [ST distance matrix between populations graphic was visualized by SplitsTree4 software v4.16.2 [p = 0.001.Moreover, the genetic distances among the studied breeds were estimated on the basis of Ariance) ; the pai v4.16.2 . SignifiGenetic variation that generates amino acid substitutions was observed in 13 codons ; the amiThe substitutions in the total sample (TOT) ranged from 0.01 to 1.00, but at the codon P240, a frequency of 0.51 for the alternative allele was estimated. At the single populations/breeds level, the highest frequencies were registered for the non-reference alleles; in particular for P194 and R215, a frequency of 0.21 in GC, and for P240, a frequency of 0.50 in BM, FL, and GC; 0.61 in FV; and 0.75 in TE. The frequency of T142 was 0.10 in the Alpine breed.PRNP analysis in the eight populations/breeds showed high polymorphic variation; among the analyzed SNPs, 13 polymorphic sites were identified giving rise to 24 alleles  and in five local populations/breeds , the codons 110 in CP and 222 in FV. The  alleles .The polymorphisms that lead to an amino acid substitution were G37V, T110P, G127S, M137I, I142M/I142T, N146S, R154H, P168Q, T194P, R211Q, Q215R, Q222K, and S240P. In addition, two silent mutations were observed at codons P42P (CCG/CCA) and S138S (AGC/AGT), but new variants compared to those already known in the literature were not observed in the analyzed sequences.The allele frequencies reported in High frequencies were observed also for the allele 1 with frequencies between 8.33% (TE) and 48.15% (AL); the allele 3 is the third most frequent. In particular, this allele is present in cosmopolitan breeds with a low percentage (9.26% in AL and 2.63% in SA), but it is rather frequent in the Italian native goats with a range between 4.17% (BM) and 12.50% . Further alleles with a frequency equal or higher of 5% are moreover observed in BM (4), CP , GC , GM , AL , and SA . Regarding the alleles common to several populations/breeds, the following have to be considered: allele 5  and allele 14 . BM is the more variable; indeed, 12 out of 24 alleles have frequencies different from zero. FST (0.058- p < 0.001). This situation can be explained by the data already discussed in The AMOVA analysis shows little variability among populations/breeds (5.83%) and a great variability within populations/breeds (94.17%), as estimated by the highly significance of the global To date, polymorphisms giving rise to the amino acid substitutions associated with resistance to scrapie in goats were investigated by many authors, and some of those SNPs were found in the populations/breeds studied in this research. In particular, S127 , able toThe H154 allele, associated with a delayed progression of the disease ,29,35, kOn the other hand, K222 allele, which confers resistance against classical scrapie ,30,33,41The Q211 allele, which seems to prolong the incubation time , is presMaestrale et al.  recordedFurthermore, double polymorphisms on codon 137 and 142 for allele 16, on codon 142 and 154 for allele 21, and on codon 110 and 142 for allele 24 were detected in a heterozygous form in the animals of our study, even if in low percentages, and these alleles were included in PHASE 2.1 output  but with a very little probability of existence. Obviously, sequencing alone does not allow determination of whether double mutations are in phase or in repulsion at the same chromosomal locus and further tests could be carried out. While a subsequent investigation of the very interesting Alpine breed samples carrying the K222 and S146 variants as markers of resistance was performed, for the other populations/breeds, the coexistence of more than one mutant allele in heterozygous form was not verified by cloning and further sequencing, so we cannot state if these alleles are in phase or in repulsion at the chromosomal locus. The reason is because this investigation was not the first instance aim of the research, but it is not excluded that it will be a future object of in-depth analysis.According to EFSA recommendations , the ranMoreover, it is possible to observe that there is no noticeable difference between the strongly selected breed SA and the other native not selected populations/breeds in terms of resistance to scrapie; at the present time, the only breed that shows S146 and K222 alleles is in fact the cosmopolitan AL breed.This work highlights that the investigated SNPs are not ubiquitously present in all the analyzed Italian goat populations/breeds and that their frequencies are different from each other; anyway, the K222 allele is present in all the populations/breeds.Taking into account also the Regulation EU 772/2020 in force, these data are a good basis to adopt scrapie eradication strategies based on genetic selection. The success achieved by the breeding programs for scrapie eradication applied to sheep represents a robust strategy to follow and adopt also in goats, where the K222 polymorphism also has the advantage of reducing susceptibility to BSE. There is indeed a large amount of experimental and in-field data relative to this allele, demonstrating and supporting a strong association with resistance to classical scrapie. On the other hand, to date, for the S146 allele, collected data indicate a strong resistance to classical scrapie but not as strong as that of allele K222. Due to the very low frequencies of alleles D146 and S146 in the rest of Europe, no data from field studies regarding resistance to other European scrapie strains are available . On thisIn order to better understand the role of all the known and unknown polymorphisms in resistance/susceptibility to the classical and atypical forms of the disease, scrapie infection studies and case reports are still necessary, also in these populations/breeds. Indeed, it is also important to establish whether these mutations have effects, either alone or in association with others.