Leishmania(L) major. As there is not any multi-epitope DNA vaccine with high efficacy against L.major, the aim of this study is to design a new multi-epitope DNA vaccine in order to have effective control upon this infectious disease through the immune bioinformatics. The L.major antigens: Gp63, LACK, TSA, LmSTI1and KMP11 were selected to design a multi-epitope DNA vaccine. The initial structure of the DNA vaccine was designed, benefiting from Gen Bank's website information. Epitopes of MHC-I antigens were predicted through the Immune Epitope Database (IEDB), and the selected epitopes were used to make vaccines construct along with linkers. New multi-epitope vaccine including 459 nucleic acids designed, and inserted between BamH1 and HindIII restriction sites of pCDNA3.1 mammalian expression vector. 12 epitopes among the chosen antigens were selected by two servers (IEDB and ANTIGEN). They had high stability and high antigenic power. Physicochemical features of vaccine measured by ProtParam server, and this structure was thermostable and hydrophilic. it’s a suitable model to study on the animal and human phases. The designed vaccine is expected to be an effective candidate through development of (CL) vaccines. However, the effectiveness of this vaccine should also evaluate in vivo model.Leishmaniasis is one of the main infectious diseases worldwide. In the midst of all the different forms of the disease, Cutaneous Leishmania (CL) has the highest incidence in the world. Many trial vaccines have been developed with the purpose of generating long-term cell-mediated immunity to  Leishmania (L), which varies from a self-healing form such as cutaneous leishmaniasis (CL) to lethal visceral leishmaniasis (VL)1. Treatment of leishmaniasis currently involves the use of a number of drugs such as pentamonial antimony, amphotericin B, miltefosine, paromomycin, which in most cases have side effects2. Vaccination is the most effective way to eradicate this infectious disease3. Structure of various types of vaccines, such as killed parasite vaccine, subunit vaccines and DNA vaccines have been studied by scientists4.There are very few Leishmania vaccines that have entered the clinical phase. Therefore, we need more studies to find a good way for the treatment5. In order to produce an effective vaccine, it is important to know the host’s immune system. To limit the CL infection, the strong produced cytokines, particularly IFN-γ, play important roles. Stimulation of T helpers, the (Th1) and (Th2) cytokines, is seen in patients with the CL infection. Among them, the Th1 has been reported to be more effective. Furthermore, the macrophages show significant capabilities in defense process. They are triggered by cytokines derived from T-cells, which are vital for controlling or aggravating the disease. Macrophages are a kind of antigen-presenting cell (APC) that distinguishes antigens of the Leishmania6.As a results, there is a critical requirement for development of a vaccine, that can bring more efficient immunity than the earlier vaccines7. Along with the various types of studied vaccines, the use of DNA vaccines has demonstrated promising outcomes. However, the use of these antigens in the form of proteins goes hand in hand by some problems, including high production price, short half-life, poor immunogenicity, low stimulation of cellular immunity, and cross-allergic reactions8. The first-generation vaccine of the CL, consists of live attenuated and killed fragmented parasites. Which is just a class of human prophylactic VL vaccine, that has entered the phase III of clinical trials until now. however, this vaccine was not successful to achieve satisfactory results9. The second-generation vaccines produced by recombinant Leishmania antigens. Among a range of approaches the LEISH-F3, a multicomponent vaccine formulated with GLA-SE adjuvant, illustrated promising results in phase I as a strong immune response inducer in healthy individuals10. More recently, a third-generation DNA vaccine that employs simian adenovirus, expressing a new synthetic gene that encode Leishmania antigens, have termed as ChAd63-KH. It has indicated potentials to be a protective and immunogenic therapeutic vaccine for the human VL, and the post kala-azar dermal leishmaniasis (PKDL) in phase I trial11. Regardless of the current progress in vaccine development, the desirable aim has not yet been attained, that is the development of a safe, effective, durable and low-cost prophylactic vaccine for human leishmaniasis12. The use of epitopes or epitope-containing peptides is advantageous, since epitopes are assessed for immune recognition and epitope specific response13. Since peptides themselves remain poorly immunogenic, the approaches that are attracting increasing interest are based on the development of peptide-based formulations in combination with potent adjuvant components 14. Peptide vaccine studies, which just a few years ago were becoming more and more marginalized, are again rising as a guaranteed finding for designing more logical vaccines15. While there is an increase in the number of new peptide-base vaccines, a major challenge is how to avoid inactivation or degradation by the immune system, and enhancing the immunogenicity of those peptides. Thus, it is essential to design vaccines using diverse approaches besides the use of other adjuvants, that can help to improve the antigen immunogenicity16.Leishmaniasis is a disease caused by a parasite of the genus L. major antigens and the selected antigens were Glycoprotein 63 (Gp63), Leishmania-activated C-kinas (LACK), Thiol-Specific Antioxidant Antigen (TSA), Leishmania major stress-inducible protein1 (LmSTI1), Kinetoplastid membrane protein 11 (KMP11), to design a new multi-epitope DNA vaccine. The protein sequences were obtained by using the corresponding Gen Bank server NCBI, (http://www.ncbi.nib.gov/genbank), which is the protein database of the National Center for Biotechnology Information. The sequence IDs of GP63, KMP11, LACK, LmSTI1 and TSA were ACL01096.2, XP003722616.1, AAA97577.1, AAB37318.1 and AAC31146.1 respectively. All sequences from diverse isolates downloaded from the Gene Bank in the FASTA format. The sequences of all antigenic proteins were checked manually and those unrelated sequences removed.We evaluated http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html and http://scratch.proteomics.ics.uci.edu/, respectively. The VaxiJen predicts the antigenicity of a protein by using a new alignment-independent method and the accuracy of this method is almost 88%. In the case of the Protein’s organism of source, a particular model selected that has a specific threshold. The VaxiJen considers a parasite protein as an antigen, when its score is more than the defined threshold which is 0.5 for the parasite data set. ANTIGENpro is a sequence-based and alignment-free predictor that has not designed for a specific pathogen group. It predicts the antigenicity of protein based on eight features sets and five machine-learning algorithms by using protein antigenicity microarray data. The accuracy of this method is near 76%.The antigenicity of the selected proteins was evaluated using two online servers, the Vaxijen and the AntigenPro at https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins.To prevent probable cross-reactivity with human proteins, the predicted CD8 + T cell epitopes screened against human proteome by NCBI BLASTp at http://tools.iedb.org/mhci/.For this purpose, H2-Dd alleles were selected as the mouse MHC-I molecules.The specific epitopes for major histo compatibility complex (MHC-I) were predicted by using immune epitope database (IEDB) server at http://bioinf.cs.ucl.ac.uk/psipred). Antigenicity Prediction of the leish21, the designed vaccine, utilized by immune epitope database server (IEDB).T-cell epitopes were linked together via gsgg, ggs, gsgs, ggssgg and ggsg linkers. The prediction of the secondary structure was performed by using an online server, the PSIPRED, available at (http://web.expasy.org/protparam).Physicochemical characteristics of the Leish21 vaccine like the molecular weight (Mw), isoelectric point (pI), instability index and the half-life, were measured by the ProtParam server available at  based on MHCI binding. Among all the predicted epitopes, only 12 epitopes with higher scores selected. We used three epitopes from each of the Gp63, KMP11 and Lack, one epitope from the LmSTI1and two epitopes from the TSA &format=png ) of mammalian expression vector.These epitopes with high binding affinity were fused together. The new multi-epitope DNA vaccine, including 459 nucleic acids synthesized. Kozak (GCCCC) is the essential site for initiation of replication. We used Alanine amino acid to improve stability of this structure Fig. , 2. The To predict the antigenicity of this structure, VaxiJen v.2 and ANTEGENpro servers were used. The prediction by the VaxiJen was 0.58 and 0.57 by the ANTIGENpro  of 15.56 KD, theoretical Isoelectric point (PI) of 6.33 and 2124 number of atoms. Additionally, the predictable half-life in mammalian reticulocytes in vitro was 4.4 h. Instability index, aliphatic index and grand average of hydropathicity (GRAVY) were 44.36, 51.09 and 0.395 respectively  was selected for further works Fig. a. The mo18. The prediction of epitopes is a significant facility for designing new vaccines4.Nowadays use of immune-informatics software, have led to save a great deal of time to design vaccine constructs. Numerous studies have carried out for epitope predictions on microorganisms such as viruses, parasites and bacteria19. Selecting several epitopes from protein antigens has several advantages over designing vaccines using full–length proteins. One of the advantages is that expressing full length of some proteins to obtain soluble protein form is difficult due to their large molecular weight, so it is better to use epitopes rather than whole protein. Moreover, some regions in the outer part of antigenic proteins have similar sequences with the host proteins and cause autoimmunity and cross- reaction with auto-antigens. Certain regions of the full-length protein also may have immunosuppressive properties. For instance, by inducing regulatory T cells, the parasite’s immune escape happens, and even leads to delay in the elimination and persistence of the parasite. By size controlling of the protein that can induce a stronger immune response, epitopes from more antigens can be used. This makes it possible to consider a large number of combinations of B cell and T cell epitopes, which can trigger a powerful humoral immune response with the help of CD4 Th cells20. So far, there is not a definite treatment report for the disease caused by L. major. The greatest strategy is vaccine development that can trigger the immune system with respect to intracellular parasite fragments and activation of TC and TH21. In addition to diminishing the intensity of reinfection, immunity against Leishmania through activating CD4+ T cells and CD8+ T cells play an important role in limiting the infection22. Multiple epitope-based vaccines have potential advantages when compared with vaccines using single or combinations of recombinant proteins. Vaccine design can be performed by selecting the most antigenic CD4+ and CD8+ T-cell epitopes from immunogenic Leishmania proteins, and therefore the avoided epitopes were those which presented low immunogenicity or detrimental immune dominance in the whole proteins23. Additionally, positives of multiple epitope-based vaccines consist of safety, low manufacturing price, easy quality control process,  in serin, can be dissolved in the hypotonic solutions. Immunogenicity of the DNA vaccines on Leishmaniasis evaluated in BALB/c mice, and the immune response provoked in animals26. Several physiological characteristics have compared based on previous studies and this study. Aliphatic index of a protein is defined as the relative volume occupied by aliphatic side chains . It may be considered as a positive factor for increasing the thermal stability of globular proteins. In a study conducted by Khatoon and Daneshi, it showed that the stability of protein against heat was much higher than other studies. In our study it has shown that the molecular weight of the design construct is lower than the protein size of other studies because in this study, fewer and more specific epitopes were used, which caused the molecular weight to be low27(Table While leishmaniasis is one of the main health issues in many countries, development of protective vaccines against it, is becoming a big concern. Peptide vaccines are potentially a new technology to struggle with the Leishmania infection. Bioinformatics procedures for the analysis of proteins have become an essential instrument for vaccine researches28. To design an effective vaccine for cutaneous leishmaniasis, bioinformatics methods, online servers, and numerous software were utilized for prediction of T cell epitops. Thus, more investigations by using in silico and in vivo patterns must be done in the future studies for assessing potency of the polytopes as a final vaccine choice.It should be noted that comparison between former studies and ours about the assessment of the designed vaccine shows that the molecular weight of the Leish21 is lower than others, and it makes an important advantage for us. Because the possibility of loop production is much lower than other vaccines. In addition, selected epitopes in this study have the highest score in the IEDB. Also, different antigens were used to design this vaccine, which this method is better than the way single-antigen vaccines are designed because it increases the efficacy of a vaccine. Aliphatic index of our vaccine has a lower quantity in comparison with the Khatoon et. al.’s, and this is because of the different molecular weight of their structures. About the vaccine-efficacy comparison with other designed vaccines, it should say that as different vaccines have positives and negatives, while they are not tested in clinical phase a proper comparison is not possible. An ideal vaccine against Leishmaniasis should have a number of potentialities, like: being immune, immunization against different types with limited number of injections for a longer duration, lower production cost, and possibility to be used both for prevent and treatmentL. major. Leish21 need to further be tested on murine models and requiring more studies on this vaccine.In this study, immune bioinformatics tools were employed to develop a new multi-epitopes vaccine which obtained from five immunogenic proteins: KMP11, GP63, LACK, LmSTI1 and TSA of the


The neutrophil-to-lymphocyte ratio (NLR) and monocyte-to-lymphocyte ratio (MLR) are biomarkers that may reflect inflammatory status in some immune-related diseases. This study aims to investigate the association of NLR and MLR with the severity and prognosis of autoimmune encephalitis (AE).A total of 199 patients diagnosed with AE in the First Affiliated Hospital of Zhengzhou University from October 2015 to October 2021 were retrospectively analyzed. The Clinical Assessment Scale for Autoimmune Encephalitis (CASE) and the modified Rankin Scale (mRS) were used to evaluate the severity of the patients at admission, and the patients were divided into mild group (CASE ≤ 4) and severe group (CASE ≥ 5) according to the CASE score. Poor prognosis was described as an mRS of 3 or more at 12 months. Binary logistic regression analysis was performed to assess risk factors for the severity and prognosis of AE.r = 0.659, P < 0.001; r = 0.533, P < 0.001) and the mRS score  in AE patients. Multivariate logistic analysis showed that NLR  and MLR  were independent risk factors for the severity of AE. In addition, the CASE score and the mRS score were positively correlated . Multivariate logistic analysis showed that the CASE at admission  and age  were independent risk factors for the poor prognosis of AE patients. The NLR and MLR at admission and whether they decreased after immunotherapy were not associated with the prognosis of AE patients (P > 0.05).NLR and MLR of severe group were significantly higher than that of mild group. NLR and MLR were positively correlated with the CASE score (NLR and MLR, readily available and widespread inflammatory markers, were helpful for clinicians to monitor disease progression and identify potentially severe patients of AE early to optimize clinical treatment decisions. BR) encephalitis have been discovered. The clinical manifestations of AE are complex and diverse, mainly including seizures, psychiatric and behavior disorders, consciousness disorders, speech disorders, autonomic nervous dysfunction, cognitive dysfunction and involuntary movements  is an autoimmune inflammatory disease targeting neuronal cell surface or synaptic proteins in the central nervous system . Dalmau ovements . Furtherovements , 5. HoweThe neutrophil-to-lymphocyte ratio (NLR) is a biomarker derived from blood routine to reflect the inflammatory status of the body. Previous studies on NLR were mostly related to tumors. A Meta-analysis in Canada showed that cancer patients with high levels of NLR tend to have a poor prognosis . NLR is The Clinical Assessment Scale for Autoimmune Encephalitis (CASE) is a new assessment scale designed specifically by Lim et al.  for AE iThe monocyte-to-lymphocyte ratio (MLR) is a new inflammatory marker similar to NLR discovered in recent years. A Korean study on endometrial cancer displayed that high levels of MLR are prominently associated with cancer recurrence and cancer-related death . InteresThis study included 199 patients diagnosed with AE in the First Affiliated Hospital of Zhengzhou University from October 2015 to October 2021. This study was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University and followed the Declaration of Helsinki. The patients included in the study fulfilled the criteria were as follows: (1) met the diagnostic criteria for AE proposed by Graus et al. , neutrophils, monocytes and lymphocytes. NLR = neutrophils/lymphocytes; MLR = monocytes/lymphocytes. The routine blood tests were performed in all patients within 24 hours after admission and before immunotherapy. Patients were followed up and routine blood tests were obtained after immunotherapy. The median duration of follow-up for routine blood tests was 27 days.The primary endpoints were disease severity (as assessed by the CASE and mRS) at admission and prognosis (as assessed by the mRS at 12 months). The CASE scale is divided into nine items, including seizures, memory dysfunction, psychiatric symptoms, consciousness, language problem, dyskinesia/dystonia, gait instability and ataxia, brainstem dysfunction, and weakness. Brainstem dysfunction included gaze paresis, tube feeding, and ventilator care due to central hypoventilation. The total scores of the CASE are 27 points. All patients were divided into mild group (CASE ≤ 4) and severe group (CASE ≥ 5) according to the CASE score at admission. The scale was independently evaluated by two neurologists who were unaware of the diagnosis through studying the detailed medical records described by the neurologists and nurses, retrospectively. Poor prognosis was described as an mRS of 3 or more at 12 months.M, IQR) and compared by Mann-Whitney U test between two groups and Kruskal-Wallis H test among three groups. Bonferroni correction was used for multiple comparisons among the three groups. The chi-square test was used to compare categorical variables expressed as numbers or percentages. Spearman correlation analysis was used to test the correlation of NLR and MLR with the disease severity. Receiver operating characteristic (ROC) curve was used to evaluate the power of NLR and MLR in predicting the severity of AE, and the area under the curve (AUC) was calculated. Logistic regression was used to analyze risk factors for the severity and prognosis of AE. The level of significance was defined as P < 0.05; The significant of Bonferroni correction pairwise comparison was P < 0.017.All data were statistically analyzed using SPSS version 26.0 . Normally distributed continuous variables were defined as mean ± standard deviation (SD), and independent samples t-test was used for comparison between two groups. Non-normally distributed continuous variables were defined as median and interquartile range . The age distribution and clinical manifestations of different subtypes of AE are remarkably heterogeneous (P < 0.017). In this study, we observed that the proportion of seizures in NMDAR encephalitis (42.0%) was significantly lower than that in GABABR encephalitis (71.8%)(P < 0.017). Consciousness disorders was more common in GABABR encephalitis (61.5%) compared with other subtypes (both P < 0.017). The proportion of psychiatric and behavior disorders (46.3%) and speech dysfunction (24.4%) were lower in LGI1 encephalitis compared with NMDAR encephalitis and GABABR encephalitis (both P < 0.017). The CASE score and mRS score of patients with LGI1 encephalitis were lower than that of patients with NMDAR encephalitis and GABABR encephalitis, with statistically significant differences in mRS score (P < 0.017). The proportion of severe patients with LGI1 encephalitis was significantly lower than that with NMDAR encephalitis (P < 0.017). There were no significant difference in gender, WBC, neutrophils, lymphocytes, monocytes, NLR and MLR among this three subtypes (P > 0.05).A total of 199 patients with AE were enrolled in this study, including NMDAR encephalitis (59.8%), LGI1 encephalitis (20.6%), and GABAogeneous Table 1.P < 0.05). There was no statistical difference in age, gender, and time from first symptoms of disease to hospitalization between the two groups. Further analysis showed that in NMDAR encephalitis, WBC and neutrophils in severe group were higher than that in mild group, lymphocytes were lower than that in mild group (P < 0.05), and there were no statistical difference in age, gender, time from first symptoms of disease to hospitalization, and monocytes between the two groups; for LGI1 encephalitis, age, WBC, neutrophils and monocytes in severe group were higher than that in mild group (P < 0.05), and there were no significant difference in gender, time from first symptoms of disease to hospitalization, and lymphocytes between the two groups; among GABABR encephalitis, lymphocytes in severe group were lower than that in mild group (P < 0.05), and there were no statistical difference in age, gender, time from first symptoms of disease to hospitalization, WBC, neutrophils and monocytes between the two groups. Among this three subtypes, the NLR and MLR of severe group were significantly higher than that of mild group  were in the mild group and 102 patients (51.3%) were in the severe group according to the CASE score. The WBC, neutrophils, monocytes, NLR and MLR in severe group were significantly higher than that in mild group, and lymphocyte was lower than that in mild group , and subtype analysis showed that NLR and MLR were positively correlated with the CASE score in NMDAR encephalitis , LGI1 encephalitis , and GABABR encephalitis . We also found that NLR and MLR were positively correlated with the mRS score in AE patients , and subtype analysis showed that NLR and MLR were positively correlated with the mRS score in NMDAR encephalitis , LGI1 encephalitis , and GABABR encephalitis , NMDAR encephalitis , LGI1 encephalitis  and GABABR encephalitis .As shown in  = 0.001)Figure 2P < 0.001); the optimal cutoff value of MLR was projected to be 0.40, with a sensitivity of 75.5%, specificity of 75.0%, and AUC at 0.771 ; An evaluation of the diagnostic value of NLR combined with MLR (NLR +  MLR) gave an AUC of 0.840 . Further analysis of the three subtypes revealed that the AUCs of NLR at NMDAR, LGI1, and GABABR encephalitis were 0.859, 0.835, and 0.756; the AUCs of MLR at NMDAR, LGI1, and GABABR encephalitis were 0.763, 0.808, and 0.849; the AUCs of NLR combined with MLR (NLR + MLR) at NMDAR, LGI1, and GABABR encephalitis were 0.866, 0.849, and 0.849  and MLR  were independent risk factors for disease severity of AE.Binary logistic regression analysis was used to identify the risk factors of disease severity of AE Table 4.BR encephalitis. All patients were divided into good prognosis group (n = 120) and poor prognosis group (n = 36) according to the mRS score at 12 months (P < 0.05). Further analysis showed that in NMDAR and GABABR encephalitis, the age of patients with poor prognosis was significantly higher than that with good prognosis (both P < 0.05). The mRS score at admission and neutrophils of patients with poor prognosis in LGI1 encephalitis were significantly higher than that with good prognosis (both P < 0.05). The monocytes of patients with poor prognosis was significantly lower than that with good prognosis in NMDAR encephalitis (P < 0.05). Among this three subtypes, the CASE score at admission of poor prognosis group were significantly higher than that of good prognosis group. There were no statistical difference in gender, time from first symptoms of disease to hospitalization, first line immunotherapy, second line immunotherapy, WBC, lymphocytes, NLR and MLR between the two groups. Multivariate logistic regression analysis showed that the CASE at admission  and age  were independent risk factors for the poor prognosis of AE patients. Further analysis showed that age was an independent risk factor for the poor prognosis of patients with NMDAR  and GABABR  encephalitis (Some patients were lost to follow-up and had insufficient follow-up (< 12 months), and 156 patients were followed up at 12 months, including 86 patients with NMDAR encephalitis, 32 with LGI1 encephalitis, and 38 with GABA2 months Table 5.P < 0.05)(P > 0.05) on NLR and MLR, we tested whether changes in NLR and MLR after immunotherapy could predict the prognosis of patients Table 7.NMDAR encephalitis is the most common subtype of AE, mainly affecting young women. In our study, NMDAR encephalitis was dominated by younger patients, but the sex ratio was almost balanced, which is similar to previous studies in Asian regions such as China and Korea , 21. ThiLGI1 encephalitis is the second most common cause of AE after NMDAR encephalitis. It mainly occurs in middle-aged and elderly patients, with a higher proportion of males than females , 23. TheBR encephalitis were included in this study, including 26 males (66.7%) and 13 females (33.3%), with a median age of 61.5 years, consistent with previous studies  found that NLR was significantly higher in patients than healthy controls, while patients with high levels of NLR tended to be more severe (mRS) at admission compared with AE patients with low levels of NLR . A studyBR encephalitis (n = 17) were 0.84, 0.64 and 0.74, respectively. The correlation coefficient between the CASE score and the mRS score of AE patients in this study was 0.849, and subtype analysis showed that the correlation coefficients of NMDAR encephalitis, LGI1 encephalitis and GABABR encephalitis were 0.868, 0.741 and 0.778, respectively. In this study, the CASE score at admission and age were associated with the poor prognosis of AE patients. Among the three subtypes, multivariate logistic regression analysis showed that the CASE score at admission was not associated with the prognosis of AE patients, partially explained by the small sample size or confounding effects of other factors. Moreover, the correlation coefficients of NLR and MLR with the CASE score were greater than that of NLR and MLR with the mRS score except for NLR and the CASE score in GABABR encephalitis and MLR and the CASE score in LGI1 encephalitis. The CASE scale has a wider distribution than the mRS scale. The CASE scale was more sensitive than the mRS scale in assessing the severity of AE and can distinguish the severity of patients within the same mRS score.Since the CASE scale was published in 2019, it has been validated in several studies. Zhang et al.  found a Currently, antibody titers are commonly used to assess the severity of AE in clinical practice. However, its effectiveness is controversial. Butler et al.  found thThis study contains several limitations. First, this study was retrospective and only completed in a single center, with an inevitable risk of bias. Second, the data of AE antibody titers in serum and cerebrospinal fluid in this study were insufficient to verify the correlation of NLR and MLR with AE antibody titers. Third, the items of the CASE scale are complex, and the accurate CASE score at 12 months for patients cannot be obtained by telephone follow-up. In addition, given that other autoimmune diseases and infections can affect NLR and MLR, we excluded patients with concomitant disease, so the results may not be extrapolated to all patients with AE. A prospective, large-sample, multicenter studies is needed to confirm our conclusion in this study in the future.In summary, our study is the first to investigate the relationship between MLR and the severity of AE. NLR and MLR, readily available and widespread inflammatory markers, were positively correlated with the CASE score and the mRS score, that is, with the severity of AE. These findings are helpful for clinicians to monitor disease progression and identify potentially severe patients early to optimize clinical treatment decisions. Both the CASE and the mRS can be used to evaluate the severity of AE, with the CASE had greater sensitivity over the mRS and it could be used to predict the prognosis of patients. In addition, the NLR and MLR at admission and whether they decreased after immunotherapy were not associated with the prognosis of AE patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.XC: conceived and designed the study. ZL and YL: collection of data. YW: statistical analysis and interpretation. ZL: wrote the paper. All authors have read and approved the final manuscript.This work was supported by the National Natural Science Foundation of China (81701295).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Statin intolerance (SI) represents a significant public health problem for which precise estimates of prevalence are needed. Statin intolerance remains an important clinical challenge, and it is associated with an increased risk of cardiovascular events. This meta-analysis estimates the overall prevalence of SI, the prevalence according to different diagnostic criteria and in different disease settings, and identifies possible risk factors/conditions that might increase the risk of SI.P = 0.04], female gender , Asian and Black race (P < 0.05 for both), obesity , diabetes mellitus , hypothyroidism , chronic liver, and renal failure (P < 0.05 for both) were significantly associated with SI in the meta-regression model. Antiarrhythmic agents, calcium channel blockers, alcohol use, and increased statin dose were also associated with a higher risk of SI.We searched several databases up to 31 May 2021, for studies that reported the prevalence of SI. The primary endpoint was overall prevalence and prevalence according to a range of diagnostic criteria  and in different disease settings. The secondary endpoint was to identify possible risk factors for SI. A random-effects model was applied to estimate the overall pooled prevalence. A total of 176 studies  with 4 143 517 patients were ultimately included in the analysis. The overall prevalence of SI was 9.1% . The prevalence was similar when defined using NLA, ILEP, and EAS criteria . The prevalence of SI in RCTs was significantly lower compared with cohort studies [4.9% (4.0–6.0%) vs. 17% (14–19%)]. The prevalence of SI in studies including both primary and secondary prevention patients was much higher than when primary or secondary prevention patients were analysed separately . Statin lipid solubility did not affect the prevalence of SI [4.0% (2.0–5.0%) vs. 5.0% (4.0–6.0%)]. Age [odds ratio (OR) 1.33, Based on the present analysis of >4 million patients, the prevalence of SI is low when diagnosed according to international definitions. These results support the concept that the prevalence of complete SI might often be overestimated and highlight the need for the careful assessment of patients with potential symptoms related to SI.  Cannon, 1 It has been clearly demonstrated that statin therapy confers significant mortality and morbidity benefits in both the primary and secondary prevention of CVD.2 Although statins are among the most commonly prescribed drugs, non-adherence and discontinuation of statin therapy is an ongoing problem worldwide.3 The most common cause of discontinuation of statin therapy is statin-associated muscle symptoms (SAMS).5 Other possible statin-related adverse effects include neurocognitive disorders, hepatotoxicity, haemorrhagic stroke, and renal toxicity.7 These conditions may lead to discontinuation, but causality has been confirmed only for SAMS, temporary elevation of aminotransferase alanine, and newly diagnosed diabetes.6 According to the International Lipid Expert Panel (ILEP), statin intolerance (SI) is an inability to tolerate a dose of statin required to sufficiently reduce an individual’s CV risk, limiting the effective treatment of patients at risk of, or with, CVD.7 The National Lipid Association (NLA) has a wider definition, including any adverse effects relating to the quality of life and leading to the decision to decrease or stop the use of an otherwise beneficial drug.8 The Luso-Latin American Consortium (LLAC) definition of SI is similar to that of the Canadian Consensus Working Group (CCWG). It refers to an inability to tolerate ≥2 statins at any dose or an inability to tolerate increasing doses. The symptoms must not be attributable to drug–drug interactions or conditions known to increase SI.10 They indicate that symptomatic criteria include intolerable muscle symptoms  or severe myopathy, and they must appear in the first 12 weeks after initiating treatment or following an increase in dose.10Cardiovascular (CV) disease (CVD) is the leading cause of morbidity and mortality worldwide, despite continuous improvement of medical treatment, diagnosis, and risk factor control.11 In contrast with randomized controlled trials (RCTs) , cohort studies suggest that SI occurs in as many as 30% of treated patients.12 However, this is likely to be an overestimate or underestimate and in many cases, the symptoms are likely to be attributable to the nocebo/drucebo effect.11The prevalence of SI is widely debated, in part because of difficulties in identification and diagnosis, possible interaction of different risk factors, different diseases, drugs, and other clinical and demographic indices.Because of these inconsistent findings, the present meta-analysis aimed to estimate the overall prevalence of SI, its prevalence according to various diagnostic criteria, in different disease settings, and to identify possible risk factors for SI.13 A PECOS  model was used to shape the clinical question and to design the search strategy (see Table S1). The following databases were searched from inception through 31 May 2021: PubMed-Medline, EMBASE, Scopus, Google Scholar, the Cochrane Central Registry of Controlled Trials, and ClinicalTrial.gov. The following keywords were used: statin intolerance, statin toxicity, statin adverse effects, statin side effects, statin-associated muscle symptoms, SAMS, statin-related myopathy, statin-related side effects, statin-related myalgia, statin discontinuation, statin withdrawal, prevalence, occurrence rate, and frequency rate (see Table S2). In addition, the references from the selected articles and relevant review articles, and the abstracts from selected congresses: scientific sessions of the European Society of Cardiology, the American Heart Association (AHA), American College of Cardiology (ACC), NLA, and European Atherosclerosis Society (EAS) were screened for additional relevant articles. The wild-card term ‘*’ was used to increase the sensitivity of the search strategy.We followed the methods recommended by the Cochrane Collaboration and complied with the reporting standards of the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) guideline of 2020.Articles were eligible if they reported the prevalence of SI either in primary or secondary prevention and met the following inclusion criteria: (i) trials or cohorts reporting SI, (ii) at least 100 participants included in the analysis, and (iii) available criteria for SI diagnosis. Exclusion criteria were as follows: (i) studies with unclear methodologies to obtain the estimates of SI frequency, (ii) studies that investigated a statin that has been withdrawn from the market, (iii) ongoing trials (unless they reported relevant interim results), (iv) studies only investigating statin discontinuation without specifying intolerance, and (v) short follow-up (<1.5 month/6 weeks).14 For the assessment of the risk of bias in cohort studies, the Newcastle-Ottawa Scale (NOS) was used. Three domains were evaluated with the following items: (i) selection, (ii) comparability, and (iii) exposure. The risk of bias in each study was judged to be ‘good’, ‘fair’, or ‘poor’.15The search, screening, and data extraction were performed independently by two reviewers (I.B. and J.R.); any disagreements were resolved through discussion with senior investigators (M.B. and P.E.P.). Non-relevant articles were excluded on the basis of title and abstract screening. For each trial, the risk of bias was independently assessed by the same investigators using the revised Cochrane RoB2 tool involving five domains . The risk of bias in each study was judged to be ‘low’, ‘high’, or ‘unclear’.8 The ILEP definition stated that SI is an inability to tolerate a dose of statin required to reduce a person’s CV risk sufficiently from their baseline risk and could result from different statin-related side effects.7 The EAS definition focused only on SAMS: the assessment of the probability of SAMS being due to a statin considering the nature of the muscle symptoms, the elevation in CK levels, and their temporal association with statin initiation, discontinuation, and re-challenge.16 As stated by the CCWG and LLAC, SI was defined as a clinical syndrome characterized by significant symptoms and biomarker abnormalities that is documented by challenge/dechallenge/re-challenge using ≥2 statins that is not due to drug interactions or untreated risk factors for intolerance10 . Because the main outcome was not limited by the type of statin, the CCWG and LLAC criteria were not used in further analyses.The primary endpoint was the overall prevalence and the prevalence based on each of the international diagnostic criteria: NLA, EAS, and ILEP. The secondary endpoint was the prevalence of SI in groups of patients with different diseases and the analysis of the association between possible risk factors/conditions and the risk of SI. According to the NLA, SI is defined as adverse effects relating to the quality of life, leading to decisions to decrease or stop the use of an otherwise beneficial drug.Table S1) were estimated from the proportion of cases of SI and sample size using the binomial exact method (Clopper–Pearson method). An inverse variance method was used for weighting each study in the meta-analysis. For the difference of subgroup analysis, we employed post hoc analysis. To investigate the differences between groups, we used the significance test. An I2 statistic was also computed for subgroup differences.14 With the inverse variance method, when the estimated probability of the condition of a single study approaches 0 or 1, the variance of the study approaches zero, which in turn causes the inverse variance to approach infinity; subsequently, the inflated inverse variance substantially increases the adjusted weight of the study in the pooled mean, resulting in an over-contribution of the study in the final pooled estimation of the meta-analysis. Therefore, to avoid the overestimated results, we conducted the Freeman–Tukey double arcsine. The final pooled result and 95% CIs were then back-transformed and expressed as percentages for ease of interpretation. The baseline characteristics are reported as the median and range. The mean and standard deviation values were estimated using the method described by Hozo et al.16 Heterogeneity between studies was assessed using Cochrane’s Q-test and the I2 index. As a guide, I2 < 25% indicated low, 25–50% moderate, and >50% high heterogeneity.17The meta-analysis was conducted using R Statistical Software , using the packages ‘meta’ and ‘metafor’ for meta-analysis. A random-effects model (DerSimonian and Laird method) was applied to estimate the pooled prevalence across the studies. The 95% confidence intervals (CIs) for the prevalence reported in the individual studies  was considered statistically significant.18Potential demographic, clinical, and drugs as modifiers of SI were further explored by meta-regression. Meta-regression coefficients and corresponding 19–194 Out of 176 articles, 112 were RCTs (195 575 patients) and the remaining 64 were cohort studies with 3 947 942 patients. The PRISMA flow diagram is shown in Figure 1 and the key characteristics of the included studies are presented in Table S3. The mean age of patients was 60.5 ± 8.9 and 40.9% were females. The White or Caucasian race made up a greater proportion of participants than Afro-American, Asian, Hispanic, or others .A total of 3569 articles were retrieved from the search after duplicates from the different databases were discarded. These articles were first screened by title and abstract, leading to 271 articles that underwent full-text review. After a stringent selection process, a total of 176 studies with 4 143 517 patients and a mean follow-up of 19 ± 7.3 months were included in the analysis.Figure S2). The prevalence based on NLA criteria was similar compared with using the ILEP or EAS definitions . The prevalence of SI in RCTs was significantly lower compared with cohort studies .The pooled prevalence of SI was 9.1% , 9.1% , respectively; 4].In an analysis stratified by the type of disease prevention, SI was more common in pooled analyses of studies which included both primary and secondary prevention . A summary of SI prevalence is shown in Figure 5. Between-study heterogeneity was large (I2 ≥ 93%). Tests assessing bias were non-significant (P > 0.28).We also compared the prevalence of SI in patients treated with lipophilic  and hydrophilic statins (pravastatin and rosuvastatin). The pooled prevalence was similar in these two types [4.0%  and female sex were associated with a higher risk of SI  . Analysis of demographic indices revealed that the prevalence of SI was associated with the percentage of participants of Asian and African-American race . However, no association was observed with White, Caucasian, and Hispanic races with SI . A summary of the meta-regression of demographic indices on SI is shown in In meta-regression analyses, age (as a continuous variable) was found to be significantly associated with the higher risk for SI [odds ratio (OR) 1.33, 95% CI 1.25–1.41; P = 0.02), diabetes mellitus , hypothyroidism , chronic liver disease , and chronic renal failure , whereas the percentage of individuals with depression was found to have a negative association with SI . Conversely, arterial hypertension was not associated with the prevalence of SI .A range of potential factors was tested for possible interaction with SI. Positive associations were found for obesity , whereas the percentage of alcohol users used showed a significant association with the prevalence of SI . Moreover, exercise , calcium channel blockers (CCB) , and antiarrhythmic agents  were associated with higher risk of SI, whereas warfarin use was not . In addition, increased statin dose was associated with a higher prevalence of SI , whereas the duration of study follow-up was not associated with the occurrence of SI . A summary of the results of meta-regression with respect to associations between risk factors and drugs on SI is shown in The percentage of smokers was not significantly associated with the prevalence of SI .The assessment of the risk of bias in the included studies using RoB2 for RCTs and NOS for cohort studies showed that most studies had moderate to high-quality level in defining objectives and the main outcomes .To the best of our knowledge, the present meta-analysis is the first to evaluate the overall prevalence of SI worldwide, the prevalence based on different diagnostic criteria and in different disease settings. The results of our meta-analysis of 176 studies with 4 143 517 patients and a mean follow-up of 19 ± 7.3 months showed that the worldwide prevalence of SI is 9.1%, irrespective of the definition applied. Older age, female gender, Asian and African-American races, obesity, T2DM, alcohol use, hypothyroidism, chronic liver, and renal diseases were associated with a higher risk of SI, as were increased statin doses and the concomitant administration of antiarrhythmic agents . There is debate on the definition of SI. We compared the prevalence of SI according to all major definitions. Despite the fact that the EAS definition of SI is focused solely on SAMS, the pooled prevalence in our analysis did not show significant differences between the EAS, NLA, and ILEP definitions.Although a wide range of values for the prevalence of SI has been reported in the literature (from 2 to 3% to as high as 50%),197–199 This also suggests that the prevalence of SI is overestimated in real-life data. It is also possible that RCTs underestimate the prevalence by excluding older patients and those with comorbidities such as chronic liver and kidney diseases and abnormal laboratory values that may increase the risk of SI. Some previous studies have reported substantially lower adherence rates in primary prevention compared with patients with CVD or after MI.200 In contrast, our sub-analysis of the pooled prevalence of SI in primary prevention (93 papers with 1 762 384 participants) and secondary prevention (54 papers with 1 166 745 participants) did not find a significant difference (8.2 vs. 9.1%). However, in observational cohort studies which included mixed patients (both primary and secondary prevention), the pooled prevalence of SI was twice as high (18%). This finding suggests that such studies overestimate the prevalence of SI. Similarly, in the subgroup analysis based on different diseases in the primary prevention cohorts  and secondary prevention , the mean overall SI prevalence was not significantly different. Likewise, regarding the safety of different classes of statins, we found no difference between lipophilic and hydrophilic statins.The prevalence of SI in cohort studies was significantly higher than that reported in RCTs. This is associated with large difficulties of correct SI diagnosis in clinical practice and lack of possibility of using of new one-of-trial approach or even cross-over design as it was applied, e.g. in PCSK9 inhibitors trials.4 It is now 20 years since the ACC/AHA/National Heart Lung and Blood Institute first identified risk factors in their recommendations for statin safety; however, there has been no attempt to validate their suggested risk factors using data from clinical trials or observational studies.201 In this meta-analysis, we have attempted to investigate what risk factors/conditions might be linked to SI prevalence using meta-regression. Pooled analysis demonstrated that many demographic, clinical, and other risk factors are associated with SI. Older age, female gender, Asian, and African-American races were associated with a higher incidence of SI, whereas White, Caucasian, and Hispanic races were not associated with higher SI risk. Many commonly observed risk factors and conditions may also be significantly associated with SI occurrence, including obesity, diabetes mellitus, hypothyroidism, chronic liver disease, and renal failure. Depression was negatively associated with SAMS, perhaps because of under-reporting in these patients.202–205 Smoking and anticoagulant drugs were not associated with SI; however, the use of alcohol, exercise, antiarrhythmic agents, and CCB was positively associated with SI. Finally, as previously reported, higher doses of statins were associated with a greater prevalence of SI.7Because statins are the gold standard for the treatment of dyslipidaemia and in the management of elevated CV risk, the most important issue during the diagnosis and management of patients with SI is the urgent need to continue statin therapy. To predict the risk of SI and to be effective in lipid management, it is critically important to know the risk factors and conditions that might increase the risk of SI.I2 = 93–99%; unknown confounding may have led to this), although this was anticipated because of the broad scope of this systematic analysis, and due to very large data, we could not test the influence analysis that would resolve the effect size of different weight across the studies. The statistical examination of potential publication bias through Egger and funnel plots is not appropriate because studies with <100 patients were excluded from this systematic review.Our meta-analysis has some limitations. Heterogeneity between studies was present in our analysis . It is also important to emphasize the importance of the nocebo/drucebo effect that was not examined in the included studies and might have distorted the final results to some extent (it might be responsible even for >50% of SAMS).The data obtained do not allow us to draw conclusions in relation to the doses of other drugs used in the included studies that could have interacted with statin therapy. Nor can we draw conclusions relating to the stage or severity of diseases such as those affecting the liver, kidney, and thyroid. Finally, our analysis cannot be used to suggest appropriate management techniques (e.g. doses of drugs and/or the severity of the diseases when statins might be used without increasing the risk of SI).Based on the data from >4 million patients, we demonstrated that the overall prevalence of SI is relatively low, especially when SI is objectively determined using the recognized international definitions. These results support the concept that the prevalence of complete SI is often overestimated and highlights the need for a very careful assessment of patients with SI, to decrease the risk of unnecessary statin discontinuation, and suboptimal lipid-lowering therapy. Clinicians should use these results to encourage adherence to statin therapy in their patients.European Heart Journal online.ehac015_Supplementary_DataClick here for additional data file.


No significant difference was found in mortality , need for ventilation , and incidence of pneumothorax  in the CPAP group compared to the ETI group. Regarding secondary outcomes, compared to the ETI group, no significant differences were found in APGAR at one minute , APGAR at 5 min , days of hospitalization , and cord pH . Conclusions: In patients with MAS, there is no significant effect of CPAP use compared to ETI on primary, specifically on mortality, need for ventilation, the incidence of pneumothorax, and secondary outcomes.Background: In neonates with meconium aspiration syndrome (MAS), continuous positive airway pressure (CPAP) may be more beneficial compared to endotracheal intubation (ETI). We evaluated the efficacy of CPAP in neonates with MAS. Methods: Four engines were used to search randomized clinical trials (RCTs). We used relative risk (RR) and mean difference (MD) with 95% confidence intervals (95%CI) to assess the effect on dichotomous and continuous outcomes, respectively. In addition, we used the Paule–Mandel (PM) random effects model due to the anticipated lack of events. Results: Three RCTs were included ( Meconium amniotic fluid aspiration syndrome (MAS) is one of the most common causes of respiratory distress in term and post-term neonates . It is aPharyngeal suctioning and tracheal suctioning improve the prognosis in neonates but do not eliminate MAS . These pA few randomized controlled trials (RCTs) have been conducted to evaluate the efficacy of CPAP versus ETI in neonates with MAS. However, the evidence has not been synthesized. Such a synthesis could aid evidence-based clinical decision-making. Because of this, our objective was to conduct a systematic review to compare the efficacy of positive pressure of ventilation (PPV) by CPAP or ETI in neonates with MAS.The protocol of the systematic review was previously published in PROSPERO CRD42018104709). Significant modifications were made in this manuscript with respect to the previous protocol. These changes were mainly directed at the main and secondary outcomes approached. The update was due to the subsequent evaluation of the evidence, which allowed the outcomes evaluated in each study to be appropriately stated. These changes did not affect the development of the protocol or the evaluation of the efficacy of the treatment compared to the control. The recommendations of the PRISMA group  were used to report this systematic review [104709. SWe searched for potential studies in Pubmed, Scopus, Web of Science, and EMBASE until 17 January 2022. We assessed randomized controlled trials (RCTs), including infants with MAS, with CPAP and ETI as the intervention and comparator, respectively. There were no restrictions on language or publishing year.Two authors (J.J.B. and M.H.G.) independently assessed the titles and abstracts of the registers. Relevant studies were chosen, and the full text was searched for further analysis. With the help of a third author, differences in selection were rectified (JCA). The studies were saved in the Endnote 20 software .The primary outcomes were mortality, need for mechanical ventilation, and incidence of pneumothorax. Secondary outcomes were incidence of pneumothorax, APGAR at one and five minutes, umbilical cord pH, and length of hospitalization (days). We used the definitions given by the authors of each RCT.Two authors (JJB and MHG) independently extracted data using a pre-developed standardized format. Disagreements were resolved by consensus between the extracting authors, and a third author (JCA) was consulted if necessary. Data extracted per study were author name, year, type of study, country, number of participants, type of intervention, type of control, birth weight, gestational age, APGAR at one and five minutes, umbilical cord pH, length of hospitalization (days), pneumothorax, need for ventilation, and mortality.The following items were assessed for each RCT with the RoB 2.0 Cochrane risk of bias assessment tool : random 2 statistic was used to assess study heterogeneity: 0–30% equaled low heterogeneity, 30–60% equaled moderate heterogeneity, and >60% equaled high heterogeneity. Because of the expected sparsity of events per arm , we additionally applied sensitivity analyses for primary outcomes using fixed-effect models and the Mantel–Haenzel method. The metabin and metacont functions from the R 3.5.1 statistics package’s meta-library were utilized.All meta-analyses used random-effects models and the inverse variance approach. We employed the PM random-effects model because of the expected lack of events. For binary and continuous outcomes, the effect was reported using relative risks (RR) and mean differences (MD) with 95% confidence intervals (95%CI). The IA total of 446 abstracts were evaluated . A total2O.The total number of individuals evaluated was 432 . All RCTThree studies ,11,12 we2 = 71%; p = 0.36; 2 = 71%; p = 0.57; 2 = 0%; p = 0.77; 2 = 98%; p = 0.31; 2= 99%; p = 0.32; 2 = 94%; p = 0.28; 2 = 0%; p = 0.79; Regarding primary outcomes, no significant differences were found in mortality between both groups  was more effective in reducing invasive ventilation requirements compared to OTI in patients with MAS . A systematic review evaluated RCTs comparing ETI and airway suctioning at birth with routine resuscitation, including oropharyngeal suctioning, in vigorous neonates, showing that ETI was not superior  [Current recommendations in the management of patients with MAS indicate being the least invasive, with the purpose of ensuring ventilation without damage to the airway anatomy and avoiding consequent collateral effects . There a86–2.60) . Taking Despite the lack of evidence, the studies addressed considered comparing the efficacy of CPAP versus ETI as ventilation measures in neonates with MAS . The goap ≤ 0.001). Regarding days of hospitalization, this study showed that patients who received CPAP had a shorter hospitalization time than intubated patients . However, the RCT by Chettri S. [p = 0.58). Nevertheless, the authors did not minimize the importance of CPAP and concluded that in the current practice of routine endotracheal suctioning for non-vigorous neonates born with MAS, it should be further evaluated.This study shows a high risk of the need for mechanical ventilation in neonates who received CPAP compared to patients who had positive pressure ventilation  and no association with the incidence of pneumothorax . However, in the relative frequency reported as a function of the need for mechanical ventilation, the proportion of patients with CPAP was lower than that in the ETI group, even though this was not statistically significant . The length of hospitalization in the group receiving CPAP was not statistically significant compared to the ETI group . Despite these differences, we decided to perform a meta-analysis because the proportions of adverse outcomes were lower in the group receiving CPAP in all studies.Different results were obtained from the RCT by Nangia S. et al. , where 1The American Academy of Pediatrics reported that the presence of meconium fluid might indicate fetal distress and an increased risk of resuscitation . HoweverAlthough it is apparently imperative to use CPAP in vigorous and non-vigorous neonates with MAS, the experimental evidence has mainly been from animal models. Consequently, Karlson K. et al.  studied Although the limited evidence on the comparison between CPAP and ETI in neonates with MAS is heterogeneous, the studies share several aspects, such as population, intervention, and control characteristics, and the results are consistent. However, the number of participants varied greatly within each study considered in this review.The main limitation in this study was the small number of RCTs; statistical heterogeneity was high, and the risk of bias in some domains was uncertain or high. These aspects may alter the interpretation of meta-analyses. In addition, a limitation of the population is that the studies did not report the severity of MAS, which could alter the analysis. However, this is the first meta-analysis to synthesize the evidence comparing both procedures, and, based on the results, we suggest new experimental and more extensive sample size studies.In patients with MAS, there is no significant effect of CPAP use compared to ETI on primary and secondary outcomes. Therefore, it is not possible to recommend its general use in clinical practice because the efficacy on primary outcomes, such as mortality, need for ventilation, and reduction in the incidence of pneumothorax, remains uncertain. In addition, more RCTs of better methodological quality and with a larger sample size are needed to learn of effects without a high risk of bias.


Patients with tic disorders (TDs) usually also have obsessive–compulsive disorder (OCD). The severity of obsessive–compulsive symptoms (OCSs) in TD is widely evaluated using the Children’s Yale–Brown Obsessive–Compulsive Scale (CY-BOCS). However, there is no survey on the severity of OCSs in patients with TD based on a Chinese sample, and the reliability and validity of the CY-BOCS in patients with TD have not been well established in China. This study examined the severity of OCSs in TDs and the psychometric properties of the CY-BOCS in Chinese pediatric patients with TD.A total of 367 patients who were diagnosed with TD [152 with Tourette syndrome (TS)] were enrolled in the Department of Psychiatry at Beijing Children’s Hospital in China. The mean age of the patients was 9.21 ± 2.06 years (range: 5–16 years). The Yale Global Tic Severity Scale (YGTSS) and CY-BOCS were used as screening tools. The psychometric properties of the CY-BOCS were assessed using Cronbach’s alpha, test-retest reliability, and construct validity.Z = −3.37, p = 0.001). However, the young adolescent group showed a higher incidence rate of OCSs than the older adolescent group (p < 0.01). Men with TDs also showed a higher incidence rate of OCSs than women (p = 0.03). The reliability and validity analyses of the CY-BOCS showed Cronbach’s alpha and test-retest reliability values of 0.81 and 0.82, respectively. The CY-BOCS showed an acceptable level in the two-factor structure (obsession and compulsive) in patients with TD. The comparative fit index (CFI) was 0.84 for TD, 0.86 for Tourette, 0.94 for the younger adolescent group, and 0.74 for the older adolescent group.The OCSs in TDs were 3.93 ± 5.15 based on the CY-BOCS in the whole sample. The older adolescent group showed higher scores than the young adolescent groups (More OCSs were identified in the TS group and the older adolescent group with TDs. The CY-BOCS showed good psychometric properties in children and adolescents with TD, especially in younger patients with TD. OCSs might be associated with age and functional impairment of TD. Tic disorder (TD) is a neuropsychiatric disorder that is characterized by recurrent tics, namely, sudden, rapid, non-rhythmic movements, or phonic productions. In our previous national survey of children’s mental disorders, the prevalence of TDs in China was 2.5% . The thrIt should be noted that when compared with other chronic motors or vocal TDs and provisional TDs, patients with TS often have more comorbidities, and these comorbidities make treatment more difficult . MoreoveThe Children’s Yale–Brown Obsessive–Compulsive Scale (CY-BOCS) is the most widely used instrument in children and pediatric OCD studies . EuropeaIn addition, previous studies have demonstrated the importance of age in TDs . For exaTherefore, this study examined the reliability and validity of the CY-BOCS in patients with TDs. We calculated the test-retest reliability, Cronbach’s alpha, and discriminant validity. Moreover, we also used confirmatory factor analysis (CFA) to test the structure of the CY-BOCS.All participants enrolled were from the Department of Psychiatry at Beijing Children’s Hospital in China from 1 October 2019 to 1 January 2020. The inclusion and exclusion criteria were age 5–16 years, meeting the diagnostic criteria for TDs according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), no central nervous system diseases, and no intellectual disability. These patients were all first onset of TDs and drug naïve, and patients diagnosed with different comorbidities were allowed.The Ethics Committees of Beijing Children’s Hospital of Capital Medical University  approved this study, and written informed consent was obtained from the parents or guardians of the enrolled children and adolescents.The CY-BOCS is a semistructured scale rated by a clinician or trained interviewer to measure the severity of obsessive and compulsive behaviors during the previous week in patients with OCD aged 8–16 years. The obsessions and compulsion subtotals are derived by adding five items  related to obsessions (range: 0–20) and compulsions (range: 0–20), respectively. The total score is the sum of the obsessions and compulsion subtotals. The CY-BOCS score and the severity of OCS are positively correlated. The cutoff point of CY-BOCS is 8 . The different severity levels of CY-BOCS (both obsessions and compulsions included) are as follows: 0–7: subclinical, 8–15: mild, 16–23: moderate, 24–31: severe, and 32–40: extreme .The CY-BOCS has the same structure and items as those for the Y-BOCS. The Y-BOCS has been translated into Chinese, and its psychometric properties have been tested . TherefoThe Yale Global Tic Severity Scale (YGTSS) is a semi-structured scale that is rated by a clinician or trained interviewer. It was developed to assess tics observed within the previous week . The fivThe two raters also completed clinical assessments. The consistency coefficient between raters was 0.87. All participants were outpatients. The assessments were performed after diagnosis in the Psychiatric Department’s Psychology Assessment Room at Beijing Children’s Hospital by one of the two psychiatrists. Furthermore, a total of 30 participants were randomly selected for retesting 1 month later to verify the test-retest reliability of the CY-BOCS.t-tests were used to compare the differences between groups, such as gender and age. If the distribution of these variables was non-normal, the Mann–Whitney U test was used. Partial correlation analysis was also used to explore the age effect of OCS in TDs (controlling for the duration of illness). Second, we used Spearman’s rank correlation analyses to examine the item-total correlations of the CY-BOCS. Third, we used Cronbach’s alpha and the test-retest reliability to assess the reliability of the CY-BOCS. Fourth, the discriminant validity of the CY-BOCS was calculated using the Pearson correlation coefficients between the CY-BOCS scores and the severity scores of the YGTSS. Finally, we performed CFA of CY-BOCS using the “lavaan” package in R (version 3.5.3). Considering the age effect for OCS in TD, we also calculated the assessed CFA of CY-BOCS in different age groups. Thus, CFA was performed for the whole sample and the TS (only patients with TS), younger adolescent (age <10 years), and older adolescent (age ≥10 years) groups.Statistical analyses were performed using IBM SPSS Statistics for Windows, version 25.0 . First, Shapiro–Wilk tests were used to assess the distribution of each item, subscale (obsession and compulsion), and total score. If the distribution of these variables was normal, A total of 382 children and adolescents with TDs were enrolled, 15 of whom were excluded because they did not complete the YGTSS or CY-BOCS assessments. Finally, 367 patients were enrolled in the study. Among these patients, 152 patients were diagnosed with TS (41.42%) [100 (27.25%) with chronic motor or vocal TD and 115 (31.33%) with provisional TD]. The mean age of the patients was 9.21 ± 2.06 years (range: 5–16 years). The younger and older adolescent groups included 230 (62.67%) and 137 (37.33%) patients, respectively.p < 0.001) , compulsion , or total scores . However, we observed significant differences between younger and older adolescents in obsession , compulsion , and total scores .The Shapiro–Wilk tests used to evaluate the distributions of each item, subscales (obsession and compulsion), and total scores showed that all items, subscales (obsession and compulsion), and total scores were non-normally distributed (< 0.001) . Therefor = 0.17, p = 0.001), compulsions , and total scores .Considering the potential influence of the duration of illness on the severity of OCS in different age groups, we used partial correlation analysis to explore the age effect of OCS in TDs while controlling for the duration of illness. The results showed a weak but significant correlation between age and the severity of obsession (p < 0.01). The TS group showed a higher incidence rate of OCS in TDs (33.55%), followed by the chronic motor or vocal TD group (28.00%) and the provisional TD group (10.43%). Second, we compared the incidence rate in different gender groups and age groups. Men with TDs showed a higher incidence rate of OCS (p = 0.03), and the younger adolescent group showed a higher incidence rate of OCS (p < 0.01). For more details, see For the whole sample, the incident rate of OCS in TDs was 24.80%. The criteria of the OCS are a CY-BOCS score greater than 7 . First, we calculated the incidence rate of OCS in different types of tic disorders . We also compared the differences among these different types (p > 0.05). In addition, the Pearson correlation between YGTSS impairment and obsession, compulsion, and total CY-BOCS scores showed significant results . Regarding the correlation between the total score of the CY-BOCS and individual items, O3, C2, and C3 showed lower correlations .The Cronbach’s alpha values for the total sample, TS group, younger adolescent group, and older adolescent group were 0.81, 0.81, 0.77, and 0.86, respectively. A total of 30 participants who were randomly selected (random number method) from the whole sample were recalled to complete the 1-month retest. The test-retest reliability of the CY-BOCS was 0.82  values of 0.84 and 0.85, respectively. The CFI values in the younger and older adolescent groups were 0.94 and 0.74, respectively , 2. The This is the first study to investigate the OCS of TDs in China based on a large sample. We found that the mean OCS in TDs was subclinical . However, more OCSs were identified in the TS group and older adolescent group with TDs. It should be noted that OCS is an important indicator of the function of patients with TDs. The age might be one of the influential factors for the OCS in TDs. Furthermore, the CY-BOCS showed good psychometric properties in patients with TDs. The CFA results showed that the two-factor structure of CY-BOCS was “stable” in younger patients (less than 10 years).Children’s Yale–Brown Obsessive–Compulsive Scale has been widely used for the assessment of OCS in TDs. Previous studies have demonstrated the fine psychometric properties of CY-BOCS in the assessment of patients with OCD , 23. ForIndeed, tic symptoms and OCS are closely related and overlap in some respects, such as epidemiology, anatomy, genetics, and treatment . Due to In the present study, items O3 and C3 showed weak associations with the total CY-BOCS. A possible reason might lie in the cultural differences between the East and the West. For example, compared with Westerners, Chinese people prefer to conceal their feelings and express fewer inner feelings . MoreoveIn the present study, OCS severity was significantly higher in the older group than in the younger group. There were three reasons for the higher OCS in the older group. First, this difference was probably due to the longer TD duration in the older group when compared with that in the younger group. Second, older patients may report OCS more clearly. Third, there was an age effect of the OCS in TDs. For example, when we controlled for the potential influence of the duration of illness, the severity of OCS still showed an association with age. This result indicated that there may be an “age effect” for OCS in TDs. For the TS groups that showed more OCS, the most likely reason was that with the persistence or the higher severity of tic symptoms, more OCS was developed. Higher levels of cognitive and metacognitive beliefs have been reported in adolescents with OCD than in children with OCD . NotablyOverall, age might have an important influence on OCS severity in patients with TDs. A higher level of severity with a lower incidence rate of OCS was identified in older adolescents with TDs. The clinical implications of these results show that the OCS should be given more attention to men with TDs. Moreover, in different types of TDs, the TS might have more OCS. Studies with larger sample sizes are warranted to confirm the effect of age and gender on OCS in TDs. For the assessment of OCS in TDs, CY-BOCS might be an important tool with good psychometric properties that can be widely used in TDs.This study had several limitations, which should be addressed in future studies. First, the main limitation of the present study is that it has no concurrent validity measures for the CY-BOCS. Due to the lack of another tool for the assessment of OCS, we could not calculate the concurrent validity of the CY-BOCS. Other scales may be better suited for the assessment of obsession and compulsion. Second, the sample only included children and not the entire age range, such as adults. Third, the sample size was small. Therefore, additional studies with larger sample sizes are needed to confirm these results. Fourth, we only focused on the OCS in TDs, while other comorbidities of TDs, such as ADHD, SIB, and anxiety, were not considered in this study. More studies are needed in the future to research the other comorbidities of TDs. In addition, we did not consider the impaired function by OCS in patients with TDs. In future studies, it would be interesting to evaluate the quality of life, cognitive profile, executive functions, and the emotional–behavioral profile of TD patients with OCS. We can also compare the differences between the TDs with and without OCS in these dimensions.More OCSs were identified in the TS group and older adolescent group with TDs. The CY-BOCS showed good psychometric properties in children and adolescents with TD, especially in younger patients with TD. OCS in TDs might be influenced by age and gender and different types of TDs. Further studies are needed to explore the OCS in TDs, especially for TS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committees of Beijing Children’s Hospital of Capital Medical University. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.YC and YL took the initiative. YG and MW finished the data collection. YL performed the data analysis. JY finished the draft. All authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intestinal metaplasia of the stomach (IM) is considered a pre-cancerous lesion and is a potential precursor to adenocarcinoma. Metabolic syndrome (MetS) has been associated with lesions to the gastrointestinal tract such as the risk of developing Barett esophagus. Vascular endothelial growth factor and leptin have been associated with either gastrointestinal tract carcinogenesis or MetS. In this context, this study was designed to analyze plasma levels of VEGF and leptin in patients with IM and MetS. Four groups of 137 participants  were created. Inclusion criteria for the presence of IM were endoscopic findings and histological confirmation, while for MetS the ATP III and IDF guidelines. Levels of plasma vascular endothelial growth factor (VEGF) and leptin (Leptin) were determined. VEGF levels were increased in IM  and IM-MetS groups . Leptin levels were found to be increased in the MetS group  and in IM-MetS . Patients with intestinal metaplasia and metabolic syndrome (I M - Me t S g r o u p) have elevated levels of VEGF, while leptin levels were associated predominantly with MetS and not with IM. IM is considered a precancerous lesion and is associated with increased risk of developing gastric cancer. Endoscopic monitoring has been proposed to check the lesion of endangered populations . HoweverIn patients with coexistence of metabolic risk factors for SD2 and cardiovascular disease  the existence of “metabolic syndrome” (MetS) has been suggested , 6. MetSStudies that have analyzed possible association of MetS and gastrointestinal tract lesions reported that patients with MetS are almost twice as likely to develop Barett esophagus (BE). The prevalence of MetS in relation to the development of BE and especially the relationship between the length of the lesion and the changes in the levels of leptin, insulin and pro-inflammatory markers, shows that the changes caused during the development of BE are continuous and are affected by the metabolic changes caused by adipokines and cytokines . On the Based on these observations we aimed to study the association between MetS and IM by analyzing markers such as VEGF and leptin which involved in both pathological conditions.VEGF represents one of the most important growth and survival factors for the endothelium. VEGF plays an important role in the regulation of angiogenesis , 15. VEGCarcinogenesis is a multistage process in which angiogenesis represents an essential step. Cancer growth, invasion and metastases depend from oxygen, nutritive substances, and growth factors delivered by angiogenesis. VEGF is one of the most important growth factors, and essential for tumor angiogenesis . SeveralLeptin is a hormone produced mainly by fat cells and helps regulate energy balance by preventing hunger, and its action was found in receptors in the arcuate nucleus of the hypothalamus. In obesity, there is a decreased sensitivity to leptin (similar to insulin resistance in type 2 diabetes), resulting in the inability to detect satiety even in large energy reserves and high leptin levels . AlthougThe study received approval from the ethics committee of GNFiliaton . The participants in the study (patients and controls) were selected from among those who underwent gastroscopy in the Gastroenterology department. According to the inclusion/exclusion criteria, those who consented to take part in the study were allocated on the basis of the findings from the clinical examination, gastroscopy, as well as laboratory and histological analyses. Those who did not have any pathological findings in the clinical/laboratory examination constituted the control group. Patients were divided in three groups. MetS (patients with MetS only), IM (patients with IM without MetS) and IM-MetS (patients with IM and MetS).Inclusion criteria for MetS (ATPIII and IDF) were abdominal obesity (AO) with an average diameter of ≥102 cm in men and ≥88 cm in women. Serum triglycerides (TRG) ≥150 mg/dL (1.7mmol/L), or treatment for elevated triglycerides. Serum HDL<40 mg/dL (1 mmol/L) in men and <50 mg/dL (1.3mmol/L) in women, or receiving treatment for decreased HDL. Blood pressure (BP) ≥130/85mmHg, or receiving treatment for arterial hypertension. Fasting glucose (Glu) levels ≥100mg/dL (5.6mmol/L), or receiving treatment for hyperglycemia.Study population included either subjects non infected, or patients infected by H. pylori, since H. Pylori has a strong association with the development of IM. However, inclusion criteria for IM was endoscopic findings and histological confirmation.Exclusion criteria were chronic inflammatory diseases , history of angiopathy (the exception was diabetics), active smokers, and PPI’s treatment. Blood sampling was carried out by an operator who had not been informed of the study. Similarly, the staff of the laboratory that carried out the analyses did not have access to the study.Blood sampling were performed before gastroscopy. All samples were taken from peripheral veins. Immediately after the blood sampling the blood was centrifuged (4000rpm for 5min) and the supernatant was stored in cryovials at -80°C.VEGF (pg/ml) levels in the patients’ plasma were determined by enzyme immunoassay , following the manufacturer’s instructions.Leptin levels (ng/ml) in the plasma of patients were identified in the method of enzyme immunoassay  following the manufacturer’s guidelines.It was estimated that at a p ≤ 0.05, to determine a 10% difference between the two groups, in terms of levels of markers, a sample of a total of 100 patients (25 patients for each group) would have a validity of 90% (GPower 3.1). A total of 137 patients were included in the study, so that a percentage of ~10% could be excluded. Statistical analysis was carried out with Statistical Package for the Social Sciences (SPSS) ver.19.0 (IBM). The regularity of the distribution of quantitative variables was analyzed by the Kolmogorov–Smirnov test. Since the levels of the variables studied had a normal distribution, they were expressed as mean ± standard deviation (SD).post hoc analysis was carried out with the Bonferroni correction. Patients’ characteristics, were analyzed with chi-square (gender) and one-way ANOVA . All analyses were two-sided, while a p < 0.05 were considered statistically significant.The differences between the groups were analyzed with one-way ANOVA and the Among the groups, no statistically significant differences were found in terms of gender and age Table 1.Analysis of MetS-related characteristics showed statistically significant differences between groups Table 2,VEGF levels were found to have statistically significant differences between groups  Table 3.Also, VEGF levels of IM-MetS were significantly higher compared to MetS (p=0.001). It appears that VEGF levels are affected by the presence of IM primarily Figure 1Leptin levels were found to have statistically significant differences between groups  Table 3.It has been demonstrated that patients with MetS are at a higher risk of developing BE even without showing symptoms of reflux . There iIn this study, both groups with IM and those with IM-MetS showed a greater increase of VEGF in plasma. VEGF has been found elevated in histological preparations of patients with stomach cancer, an increase which was parallel to the expression of COX-2 . In partThe determination of plasma leptin levels showed a significant increase in levels in patients with MetS and IM-MetS. Regarding the increased levels of leptin in patients with MetS there are several reports confirming their increase, which is mainly related to the mass of adipose tissue, while on the other hand its levels are also related to insulin resistance .In our study, patients with IM, like those with IM-MetS, did not experience elevated leptin levels. Reports from the international literature regarding leptin levels in IM patients are contradictory. It appears that IM is not related to plasma leptin levels, as patients with BE and IM, or with IM, did not experience increased levels , 39. HowThis is the first study which analyzes these markers in patients with MetS and IM, as well as with both MetS and IM. We believe that the results, although this is not a randomized trial, might contribute to conception and design of studies, in order to further elucidate the mechanisms, as well as the association between MetS and IM, especially since either MetS or IM are associated with GI carcinogenesis.This study has some limitations which must be acknowledged. Firstly, the analyses of plasma VEGF and leptin levels concerned MetS in general, but not each component of it. It would be very interesting to study the possible contribution of each component of MetS in the development of IM. Moreover, it could be interesting to study the possible association of each form of VEGF with MetS and the development of IM.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by Filiates General Hospital ethical committee. The patients/participants provided their written informed consent to participate in this study.Conceptualization, GP-G, FF, and GG. Methodology, GP-G, CT, FF, AG, KT, KV, and GG. Statistics, GP-G, FF, and KT. Investigation, GP-G, FF, GG, and KV. Data curation, AG, CT, GP-G, GG, and KV. Writing—original draft preparation: GP-G, KT, and KV. Writing—review and editing: all authors. Supervision, KV and GG. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: To investigate the combined impact of being engaged in resistance training (RT) and meeting the physical activity guidelines through sports participation (SP) on cardiovascular and metabolic parameters in lean adolescents. Methods: A longitudinal study, part of the ongoing study entitled “ABCD Growth Study” , assessed data from 64 adolescents . Metabolic and cardiovascular outcomes were analyzed as dependent variables. For the independent variables, sports participation and resistance training were considered, and for the covariates, sex, chronological age, body weight, height, and somatic maturation. Results: After 12 months of follow-up, the RT + SP presented improvements in triglycerides (TG) and the SP presented a reduction in LDL-c, TG, and glucose when compared to the control group. Conclusions: Being engaged in RT and SP is a good strategy to improve health in eutrophic adolescents, with a great impact on TG from the lipid profile. Cardiovascular diseases (CVD) represent the main cause of death in adults worldwide . In factSports participation (SP) is a subset of physical activity and constitutes the main manifestation of physical exercise during adolescence, being influenced by sociocultural aspects . EngagemFurthermore, engagement in resistance training (RT) is linked to improved sport performance and muscle strength . MoreoveAlthough there are solid guidelines recommending both SP and RT for adolescents, their combined effect on cardiovascular and metabolic aspects of adolescents is still under investigation. Pediatric exercise science has extensively investigated the impact of RT and SP on the health aspect of overweight/obese adolescents but in lean adolescents this information is unclear.Thus, the aim of this manuscript was to elucidate the features of systematic training and physical activity in adolescents with different metabolic characteristics. Additionally, to analyze the combined impact of being engaged in RT and meeting the physical activity guidelines through SP on cardiovascular and metabolic parameters in lean adolescents compared to both only meeting the physical activity guidelines through SP and no engagement in SP or RT at all.This longitudinal study was part of the ongoing study entitled “ABCD Growth Study” , which is being carried out in the city of Presidente Prudente, State of São Paulo, Brazil. The ABCD Growth Study was approved by the Ethics Committee of São Paulo State University (UNESP [process: 1.677.938]). Data collection and analyses were performed by researchers of the Laboratory of Investigation in Exercise (LIVE), which is part of the Department of Physical Education of UNESP. Parents/guardians, and adolescents signed the written consent form.The ABCD Growth Study is a pragmatic trial in which researchers are interested in the way sports participation affects health outcomes among adolescents in the “real world”, targeting improved external validity of the findings. Therefore, researchers monitor adolescents over time but do not interfere in their training routine, different from a clinical trial where the variables  are controlled in order to reach maximum internal validity  while reducing external validity.The dataset for this manuscript was collected in 2017 (baseline) and 2018 (12 months of follow-up). Further details about the sampling process can be found elsewhere . BrieflyAt baseline, 285 adolescents were assessed and started the follow-up period. After 12 months of follow-up, 189 adolescents were reassessed. The reasons for the 96 dropouts were as follows: fear of blood collection, having moved to another city, not having enough time to participate in data collection, and the desire to drop out of the study. From those 189 adolescents who remained in the study, 5 were excluded as they were 18 years old at baseline, 14 were excluded due to missing data in at least 1 of 4 lipid variables at follow-up, 1 adolescent was excluded due to missing data for anthropometry at follow-up, and 63 were excluded due to a diagnosis of obesity at baseline. From the 106 lean adolescents remaining, 42 were excluded due to not meeting the physical activity guidelines through sports participation. Finally, the sample was composed of 64 adolescents (48 boys and 16 girls) .Systolic (SBP) and diastolic blood pressure (DBP) were analyzed using an automatic device  previously validated for the pediatric population . After tAll variables were treated as absolute changes (Δ) after 12 months of follow-up .Researchers monitored adolescents over a period of 12 months, assisted by coaches and assistant coaches. Information about RT was collected through a face-to-face interview , and adolescents who reported RT at baseline and follow-up were considered consistently engaged. The heart rate of adolescents engaged in sports was assessed during two whole training sections , and time in moderate-to-vigorous physical activity was calculated  ,16. In tAdolescents who were assessed in schools and declared no engagement in sports at baseline (and over the 12 months prior to baseline) were tracked as “control” and only those who remained not engaged in both sports and RT were considered in this manuscript.Sex and chronological age were collected through a face-to-face interview. The sex was defined as the biological sex at birth. Body weight was measured using a scale  and height and cephalic trunk height using a fixed stadiometer . Body fatness was estimated by bone densitometry . Anthropometric data were used to estimate somatic maturation through the peak height velocity (PHV) .p-value < 0.05) and analyses were performed using the software BioEstat (version 5.0).Descriptive data are presented as mean, standard deviation (SD), and 95% confidence interval (95% CI). Analysis of variance (ANOVA) and covariance (ANCOVA) were used to compare the variables according to sports participation and RT in crude and adjusted approaches, respectively. Post-hoc tests were used when necessary . ANCOVA models were adjusted for sex, age, maturation, body fatness, and the baseline values of the dependent variable. Levene’s test assessed the assumption of homogeneity of variances , and measures of effect size were expressed as eta-squared (ES-r) as follows: ES-r < 0.064 , ES-r ≥ 0.064 and < 0.140 (moderate effect size), and ES-r ≥ 0.140 (high effect size). Statistical significance was set at 5%  and the sport-RT group . Similarly, the PHV of the sport group was lower than the control group  and the sport-RT group , although adolescents in all groups had passed the peak. The glucose level was lower in the control group than the sport-RT group .Baseline characteristics of the sample, stratified according to groups, are presented in p-value = 0.033). In parallel, when the data were divided by SP, there were differences for LDL-c, TG, and glucose .After 12 months, the control group presented a significant increase for LDL-c (6.8 mg/dL [0.45 to 13.1]), TG (16.0 mg/dL [7.0 to 24.9]), and glucose (4.2 mg/dL [0.9 to 7.5]), which was higher than observed for the sport group  and sport + RT group (TG) .ANCOVA identified that even after controlling for variance explained by the confounding factors, adolescents who were simultaneously engaged in RT and SP presented lower TG levels (−7.1 mg/dL [95% CI: −22.1 to 7.9]) than the control group (15.3 mg/dL [95% CI: 5.5 to 25.2]). The magnitude of the difference was moderate. Moreover, differences for LDL-c and glucose did not remain significant in the adjusted models, but the effect size observed for LDL-c was of moderate magnitude .This pragmatic trial identified that simultaneous engagement in both sports and RT was significantly related to moderate improvements in TG.The first aspect of our findings is the fact that, from the seven outcomes investigated, only one was significantly related to the combined engagement in sports and RT. In fact, this is not a surprise, mainly because the adolescents assessed were non-obese, and thus, abnormal values are less common . HoweverTo our knowledge, there are limited data investigating the combined impact of RT and sports participation on cardiovascular and metabolic parameters in lean adolescents. Our main findings regarding the combined impact of both on lipids are in agreement with other studies assessing sports participation ,22, but In terms of glucose parameters, differences became non-significant on the adjusted model. The absence of significant differences for glucose was attributed to differences at the baseline (which were higher in the group engaged in both SP and RT), although no abnormal values were observed for glucose in the sample (glucose > 126 mg/dl ). TherefThe background supporting the use of exercise to treat obese adolescents is consistent. Data provided by a randomized clinical trial indicate that the impact of aerobic exercise combined with strength training in overweight and obese adolescents is one of the best strategies to achieve recommended lipid profile values . In addiThe limitations of the present study should be recognized. First, the absence of training parameters for RT  is relevant because, without these data, it is not possible to describe in depth how RT was administered to these adolescents. Second, the pragmatic approach adopted in this study provides a better inference of these findings in the reality of these adolescents’ lives. In fact, the combination of different sports gives a “real-world” to our findings but limits the possibility of describing which sport would be more beneficial for the metabolic health of lean adolescents, if any. Finally, the reduced sample size, especially in the group SP + RT, limits sex-specific analyses.In summary, combined engagement in sports of moderate-to-vigorous intensity and RT seems a relevant strategy to improve lipid profiles in lean adolescents.