Is cerebrospinal fluid (CSF) diversion associated with improved outcomes or intracranial pressure in children with severe traumatic brain injury (TBI)?In this comparative effectiveness study of 1000 children with severe TBI, there was no association between CSF diversion and Glasgow Outcome Score–Extended for Pediatrics at 6 months after injury in propensity-matched participants. However, CSF diversion was associated with decreased intracranial pressure in the propensity-matched participants.These findings suggest that the current evidenced-based guidelines that support CSF diversion as a first-line therapy for TBI in children should be reconsidered. This comparative effectiveness study assesses the associations of cerebrospinal fluid diversion with Glasgow Outcome Score–Extended for Pediatrics and decreased intracranial pressure in children with severe traumatic brain injury. Diversion of cerebrospinal fluid (CSF) has been used for decades as a treatment for children with severe traumatic brain injury (TBI) and is recommended by evidenced-based guidelines. However, these recommendations are based on limited studies.To determine whether CSF diversion is associated with improved Glasgow Outcome Score–Extended for Pediatrics (GOS-EP) and decreased intracranial pressure (ICP) in children with severe TBI.This observational comparative effectiveness study was performed at 51 clinical centers that routinely care for children with severe TBI in 8 countries  from February 2014 to September 2017, with follow-up at 6 months after injury . Children with severe TBI were included if they had Glasgow Coma Scale (GCS) scores of 8 or lower, had intracranial pressure (ICP) monitor placed on-site, and were aged younger than 18 years. Children were excluded if they were pregnant or an ICP monitor was not placed at the study site. Consecutive children were screened and enrolled, data regarding treatments were collected, and at discharge, consent was obtained for outcomes testing. Propensity matching for pretreatment characteristics was performed to develop matched pairs for primary analysis. Data analyses were completed on April 18, 2022.Clinical care followed local standards, including the use of CSF diversion (or not), with patients stratified at the time of ICP monitor placement (CSF group vs no CSF group).The primary outcome was GOS-EP at 6 months, while ICP was considered as a secondary outcome. CSF vs no CSF was treated as an intention-to-treat analysis, and a sensitivity analysis was performed for children who received delayed CSF diversion.P = .08), but there was a decrease in overall ICP in the CSF group .A total of 1000 children with TBI were enrolled, including 314 who received CSF diversion  and 686 who did not . The propensity-matched analysis included 98 pairs. In propensity score–matched analyses, there was no difference between groups in GOS-EP (median [IQR] difference, 0 [−3 to 1]; In this comparative effectiveness study, CSF diversion was not associated with improved outcome at 6 months after TBI, but a decrease in ICP was observed. Given the higher quality of evidence generated by this study, current evidence-based guidelines related to CSF diversion should be reconsidered. CSF diversion, was first described in 1774,2 and it remains one of the most common neurosurgical procedures for neurological conditions, including traumatic brain injury (TBI).3 Removing CSF can lead to decreases in intracranial pressure (ICP),4 and uncompensated increases in ICP can impair cerebral perfusion and lead to adverse outcomes. Placement of devices to drain CSF can allow for ICP measurement at the bedside, while intraparenchymal ICP catheters are also available to measure ICP without performing CSF diversion. The risk-benefit decision for which device to place is central to contemporary neurocritical care.Drainage of cerebrospinal fluid (CSF) for neurosurgical emergencies, called 8 Subsequently, 7 versions of evidenced-based guidelines for severe TBI for adults12 and children15 suggest use of CSF diversion. The current recommendation for children is that that CSF drainage through an external ventricular drain (EVD) is suggested to manage increased ICP based on data from 56 children in 3 published reports.15 Because of the weakness of the evidence,16 we sought to determine the associations between CSF diversion and neurological outcomes in children with severe TBI. After conducting a survey of 32 pediatric centers to determine their current practices related to CSF diversion, we found substantial intercenter and intracenter variability, strongly suggesting a lack of equipoise that would imperil a randomized study design.17 Therefore, we conducted an observational comparative effectiveness study using propensity matching to test the primary hypothesis that CSF diversion is associated with improved outcomes after severe TBI. As a secondary hypothesis, we sought to determine if CSF diversion in these matched patients is associated with decreased ICP.Seminal studies demonstrated associations between intracranial hypertension and mortality after TBI, and these studies concurrently led to recommendations for CSF diversion as a treatment strategy.STROBE) reporting guideline.The Approaches and Decisions for Acute Pediatric TBI (ADAPT) trial was an observational comparative effectiveness study funded by a cooperative agreement with the US National Institute of Neurological Disorders and Stroke). The study included sites in the US, United Kingdom, Spain, the Netherlands, India, South Africa, Australia, and New Zealand  score of 8 or less at the time of monitor placement. Because we intended ADAPT to inform the evidenced-based guidelines for severe TBI and many of the guideline topics are related to ICP-based therapies, we limited inclusion to children who had ICP monitors placed as part of their clinical care. Importantly, sites chose the ICP monitoring modality  at their discretion. We recorded the GCS score at the time of ICP monitoring to ensure that all participants met the current definition for severe TBI (GCS score ≤8). Exclusion criteria were pregnancy  and ICP monitor placement at another institution (to ensure data availability for early therapies administered).20 Patient demographics, injury details, imaging findings, severity of illness scores, and prehospital and resuscitation events were collected consistent with the National Institute of Neurological Disorders and Stroke TBI Common Data Elements (CDE)23 , resuscitation phase , ICP therapy phase , hospital phase , and follow-up phase (defined as 6 months after ICP monitor placement time), as previously reported.Sites recorded information regarding all neurosurgical procedures that occurred, including insertion of an EVD. Sites recorded the strategy of CSF diversion (CSF diversion group vs no CSF diversion group) at the time of ICP monitor placement and this strategy was used as an intention-to-treat decision for this analysis (eTable 2 in t test (or Wilcoxon Rank Sum test) or χ2 test (or Fisher exact test). Bivariate regression  models were used to assess the strength of the association of CSF diversion with each outcome . A concern with observational studies is that there are confounders and biases associated with treatment allocation . Propensity score analysis was used to reduce the potential impact of confounding and selection bias effects. The propensity score is the probability of treatment assignment conditional on observed baseline characteristics. We estimated the propensity scores using the generalized boosted models (GBM) approach26 and considered all characteristics collected. We used the Toolkit for Weighting and Analysis of Nonequivalent Groups (twang) software package (RAND)27 and SAS macros (SAS Institute) to estimate and evaluate the propensity scores. Propensity-matched analysis with a 1:1 ratio was used to compare the outcomes of interest. We allowed a maximum of 3 splits for each tree in the model, allowing for 3-way interactions among all covariates to be considered. The shrinkage parameter was set to 0.005 to ensure a smooth fit. Balance tables and plots were used to assess the quality of the propensity scores, to compare the propensity score distributions between treatment groups, and to evaluate the common support. From the GBM approach, a propensity score indicating the probability of CSF diversion strategy (yes or no) given observed baseline characteristics was obtained for each participant. Caliper matching without replacement within specified calipers of the logit of the propensity score was used for propensity score matching.29Baseline characteristics were summarized for each group and compared with independent sample 30 After propensity matching, a conditional logistic regression model was used to assess the association of propensity-matched CSF group with mortality and complications. A conditional Cox proportional hazards model was used to assess the association of propensity-matched CSF group with time to death. Baseline characteristics that were unbalanced after matching by propensity score were included in the models. As a sensitivity analysis, a combination of inverse probability of received treatment weighting and unbalanced covariate adjustment  was performed if the propensity scores were not able to completely balance participants’ characteristics for all outcomes. A similar analysis was performed as a sensitivity analysis to assess the effect of including children with delayed CSF diversion in the CSF diversion group. In an effort to broaden the number of participants included in the matched analysis, an additional sensitivity analysis was conducted excluding a subsample of participants not likely to receive the intervention. A classification tree model was used to identify subgroups who were not likely to receive CSF diversion. The independent variables in the classification tree were those that were included in the primary propensity score model. A propensity model was then fit using the independent variables included in the propensity score from the primary analysis. This analysis also resulted in a limited number of matched pairs. To increase the area of common support and number of matched pairs, the propensity model was restricted to characteristics that were associated with both the outcome and the exposure.The primary outcome of the study was GOS-EP scores at 6 months. Other end points were evaluated as exploratory, and no adjustments for multiple comparisons were made. For our primary outcome of propensity-matched participants, the signed rank test was used to test the association of group with GOS-EP score. An ordinal regression model was then used to estimate the association of CSF diversion with GOS-EP score after controlling for characteristics not balanced through matching.P = .05. All analyses were conducted using SAS software version 9.3 (SAS Institute). Data were analyzed from [placeholder] to [placeholder].A longitudinal mixed effects regression model was used to compare the mean ICP over time between groups. The model included a main fixed effect for and indicator of CSF diversion therapy and a random effect for matched pair. All tests were 2-sided, and the significance level was set at A total of 1018 children were screened for inclusion, and 18 children were excluded for having an ICP monitor placed outside of a study site . A totalP = .96). There was no difference for death at 28 days  or time to death  in adjusted analysis or after adjusting for characteristics not balanced after propensity matching   (P = .09)    . The lacP = .12) . Kaplan-P = .12) . A totalIn this comparative effectiveness study, the most comprehensive analysis of children with severe TBI to our knowledge, there was no association of CSF diversion with improved overall outcomes at 6 months after injury. However, in an exploratory analysis, we found an association between CSF diversion and lower ICP. These findings may inform future recommendations for CSF diversion for children with severe TBI.32 published a case series of 22 children with CSF diversion, reporting 22% mortality. However, the intent of this early report was to demonstrate the utility of the pressure-volume index, rather than evaluating the effectiveness of CSF diversion. A 2008 study in 23 children by Jagannathan and colleagues33 reported 13% mortality and observed that most survivors achieved ICP control. A 2011 case series by Andrade and colleagues34 reported outcomes of 11 children with continuous CSF diversion, but comparisons between CSF diversion strategies were not performed.CSF diversion has been used in children with severe TBI for decades, yet the recommendations supporting its utility are based on limited studies. Shapiro and Marmarou35 compared the effectiveness of continuous vs intermittent CSF diversion in 62 participants (31 participants per group) and found that the continuous CSF diversion group had improved ICP (5.66 mm Hg lower than the intermittent group) and fewer episodes of intracranial hypertension. However, a comparison between diversion groups was not performed. A study by Griesdale and colleagues36 compared 93 adults with CSF diversion vs 73 adults without this therapy in an uncontrolled observational study. CSF diversion was associated with increased mortality; however, subgroup analysis of patients with GCS scores less than 6 showed some benefit, leading to one of the aforementioned recommendations stated.12 A secondary analysis from 2019 by Bales et al37 assessed outcomes in the citicoline brain injury treatment trial based on CSF diversion utilization. CSF diversion was performed at the discretion of the site, and a comparison between participants who did and did not receive CSF diversion  was performed. Bales et al37 found that CSF diversion was associated with higher mortality and worse neuropsychological outcomes. The findings of our exploratory analysis related to ICP, which was able to control for covariates and match patients based on relevant characteristics, suggest that CSF diversion could be useful to control intracranial hypertension early after severe TBI in children, as some of these smaller adult studies have indicated.CSF diversion is also recommended for adults with severe TBI, with recommendations that continuous CSF diversion may be considered to lower ICP burden and CSF diversion may be considered within the first 12 hours for patients with GCS scores less than 6 to lower ICP. The studies supporting these recommendations have similar limitations to those that inform the pediatric guidelines. A study by Nwachuku and colleaguesOur study represents the highest quality of evidence regarding CSF diversion after TBI to out knowledge. Although our study design could only detect associations between the therapy and outcomes and could not discern cause and effect relationships, this method may be the most feasible way to test the effectiveness of this therapy that is already part of contemporary practice. A randomized clinical trial would be a superior method to determine the effectiveness of CSF diversion, if such a study could be accomplished. However, several factors could hamper such a study. Since we did not demonstrate a beneficial association of CSF diversion with overall outcome, estimating an effect size to plan a randomized trial would be challenging. Equipoise regarding CSF diversion may also be negatively impacted by our primary finding related to overall outcome, but our exploratory analysis related to ICP may support such an approach.This study has some strengths as well as limitations. As the largest prospectively enrolled study in this population to our knowledge, we were able to adjust for many measured covariates that might affect outcomes, including more than 1200 unique data elements. However, unmeasured covariates could influence our findings. Our enrollment procedures allowed us to minimize selection bias by including consecutive participants, but these methods limited our ability to obtain outcomes from all participants. This limitation was mitigated to some degree by our follow-up procedures. Our broad inclusion criteria and consecutive enrollment procedures intentionally included children who are often not included in clinical trials, particularly those with penetrating injuries and with abusive head trauma. While this limits the direct comparison of our study with some others, we thought it was important to determine the overall effectiveness of CSF diversion in the entire pediatric TBI population. Despite our large sample size, we were unable to stratify participants into subgroups  that might have demonstrated some benefit. While we were able to perform sensitivity analyses, we were unable to evaluate if children with posttraumatic hydrocephalus would benefit. Additionally, while the study included 1000 participants, the participant characteristics and statistical methods yielded a limited number of participants for the primary analysis, which may limit the generalizability of the results.This comparative effectiveness study did not find a beneficial association of CSF diversion with GOS-EP at 6 months after severe TBI in children. Further studies will be necessary to determine if there are subsets of children who might benefit. However, in light of our findings, recommendations for CSF diversion may need to be reconsidered.


Development of non-invasive treatments such as pharmacological agents is hampered by a lack of suitable models and incomplete understanding of the underlying pathophysiology. This Research Topic aimed to collect emerging evidence and novel perspectives on CSF dynamics and ICP elevation in neurological disease to highlight new developments in this field.Cerebrospinal fluid (CSF) provides a protective cushioning to the brain and the spinal cord, and serves an important role in nutrient transport and waste removal, essential for maintaining normal neuronal function. Homeostatic feedback mechanisms are responsible for the strict regulation of CSF formation, composition, volume, turnover, and flow  dysfunction as primary events in the pathophysiology of IIH. This suggests that BBB dysfunction and astrogliosis , which are highly expressed in astrocytic endfeet (AQP4) and choroid plexus epithelial cells (AQP1), in the pathophysiology of idiopathic normal pressure hydrocephalus, and outlined new targets for further research. Overall these studies and reviews demonstrate the important role played by the cells of the neurovascular unit in the pathophysiology of these diseases, and the need for further studies to determine their contribution to disease pathology. Other reviews in this topic discuss the role of the choroid plexus epithelium in neurological disease  and the role of ependymal cilia in hydrocephalus .Traditional views of CSF dynamics and ICP continue to be challenged as the field continues to evolve and new theories are formed, and novel models developed to study these complex mechanisms. Indeed, Hornkjøl et al. and Perera et al. have presented novel findings on CSF dynamics from a computational model and a non-invasive magnetic resonance imaging (MRI) technique respectively. Hornkjøl et al. demonstrated the importance of CSF dynamics on transport and clearance of substances, not only in the spinal subarachnoid space, but also deep into the brain. Research on the ‘glymphatic system’, a proposed waste clearance system of the brain, has mainly focused on fluid flow and transport within the brain  MRI approach to determine BCSFB function in healthy and hypertensive rats. The findings suggest that BCSFB could be a key site vulnerable to systemic hypertension, leading to changes in CSF homeostasis in hypertensive subjects.Finally, Overall, this Research Topic provides a cross-cutting perspective of CSF dynamics and ICP elevation, and showcases a range of thought-provoking approaches and models to study the complex underlying mechanisms. The novel discoveries presented here will be exciting to follow as the field moves forward from traditional concepts of CSF dynamics.Conceptualization and writing—original draft: AP. Writing—review and editing: AP, VV, and MS. All authors approved the submitted version.


O diagnóstico de síndrome coronária aguda (SCA) e a estratificação de risco contemporâneos são fundamentais para o manejo apropriado e redução da mortalidade e eventos isquêmicos recorrentes, tanto na fase aguda quanto após hospitalização. A Definição Universal de Infarto do Miocárdio recomenda a detecção de curva de troponina acima do limite superior do percentil 99. Avaliar a ocorrência de óbito e infarto agudo do miocárdio (IAM) na fase precoce em pacientes sem elevação de troponina , pacientes com mínima elevação [acima do percentil 99 ], e pacientes com maiores elevações [acima do ponto de corte para IAM pelo kit utilizado ]; e avaliar o impacto dos níveis de troponina na indicação de estratégia invasiva e revascularização miocárdica. Estudo de corte transversal de pacientes com SCA com avaliação do pico da troponina I, escores de risco, análise prospectiva de desfechos clínicos até 30 dias e testes bilaterais de significância, com nível de significância adotado sendo < 0,05. Foram avaliados 494 pacientes com SCA. Troponina > percentil 99 e abaixo do ponto de corte, assim como valores maiores (acima do ponto de corte), foram associados à maior incidência do desfecho composto  e de revascularização percutânea ou cirúrgica , sem diferença significante em mortalidade até 30 dias. Valores de troponina elevados acima do percentil 99 pela Definição Universal de IAM apresentam papel prognóstico e agregam informação útil ao diagnóstico clínico e escores de risco na identificação de pacientes com maior probabilidade de benefício com estratificação invasiva e procedimentos de revascularização coronária. Por outro lado, alguns estudos não encontraram associação entre níveis altos de troponina e piores desfechos clínicos.18Fatores como a gravidade e a complexidade da DAC, o uso prévio de ácido acetilsalicílico e a precocidade da angiografia coronária estão associados ao pico da troponina na SCASSST.International Federation of Clinical Chemistry e da National Academy of Clinical Biochemistry, troponina elevada é definida acima do percentil 99 para uma população considerada saudável e com coeficiente de variação (CV) intraensaio < 10%,19 embora muitos kits de troponina apresentem imprecisão analítica com base nesse percentil.22 Portanto, para o uso da troponina no diagnóstico de IAM, é necessária uma curva ascendente e/ou descendente do biomarcardor, com pelo menos um valor acima do percentil 99, baseado na população de referência e conforme sexo, etnia, e outros fatores.24 Alguns estudos ressaltam a importância de uniformizar o nível de troponina para diagnóstico de IAM em laboratórios hospitalares para aprimorar a decisão clínica, individualizar o valor para população atendida naquele hospital, além de facilitar relatórios em estudos clínicos.26Conforme recomendações da Desse modo, os objetivos deste estudo foram avaliar a ocorrência de desfechos clinicamente relevantes  em pacientes com SCASSST na fase precoce; comparar três grupos divididos de acordo com respectivas faixas de mensurações de troponina I: sem elevação , com mínima elevação , e com maior elevação [“Ponto de corte de maior acurácia” estipulado no kit local ; e avaliar a associação entre os três grupos e a indicação de estratégia invasiva e de procedimentos de revascularização miocárdica na fase hospitalar. A hipótese é de que o nível do percentil 99, mesmo abaixo do valor de corte do kit de troponina I esteja associado a impacto clínico e maior indicação de estratificação invasiva e revascularização miocárdica, em comparação com níveis negativos, corroborando a proposta da definição universal para diagnóstico de IAM.Estudo observacional, transversal, com seguimento até 30 dias para avaliação prospectiva de óbito, infarto, e desfecho composto em pacientes com SCASSST admitidos em Unidade Coronária (UCO), separados em grupos conforme nível de troponina. Todos os eventos clínicos foram pré-especificados e avaliados após coleta sistemática de informações em banco de dados, além das indicações de estratificação de risco invasivo ou não invasivo, tratamentos intra-hospitalares, e testes laboratoriais de rotina. Consentimento informado foi obtido por meio de termo de consentimento livre e esclarecido. Este estudo recebeu aprovação pelo Comitê de Ética em Pesquisa local em abril de 2019. O recrutamento dos pacientes ocorreu entre maio de 2019 e janeiro de 2020.• Idade ≥ 18 anos•  Hospitalizados na UCO•  Diagnóstico de SCASSST• O diagnóstico de IAMSSST obedeceu a dois dos seguintes critérios:- Quadro clínico sugestivo de SCA;- ECG demonstrando depressão do segmento ST, inversão de ondas T ou achados inespecíficos.- Curva de troponina, ascendente e/ou descendente, com pelo menos um valor acima de 0,12 ng/mL .O reinfarto foi definido conforme recomendações da 4ª definição universal de IAM, sendo suspeito na presença de sinais ou sintomas de infarto, necessitando de nova dosagem de troponina. Um aumento de 20% nos níveis de troponina em pacientes já com elevação de troponina, ou um novo aumento de troponina em pacientes com exame previamente normal, confirmam o diagnóstico.  • O diagnóstico de angina instável obedeceu a dois dos seguintes critérios:- Quadro clínico sugestivo de SCA;- ECG demonstrando depressão do segmento ST, inversão de ondas T ou achados inespecíficos.27e Diretriz da Sociedade Europeia de Cardiologia28).- Ausência de troponina com valores acima de 0,034 ng/mL  e escores de risco GRACE e CRUSADE. Os testes laboratoriais, procedimentos e tratamentos na fase hospitalar seguiram protocolos da instituição.Ortho Clinical Diagnostics, com valor de percentil 99 de 0,034 ng/mL, e ponto de corte para diagnóstico de IAM de 0,12 ng/mL, para sensibilidade de 95% e especificidade de 93% . As variáveis categóricas foram expressas como frequências e porcentagens, e comparadas por teste do qui-quadrado ou teste exato de Fisher. A análise dos desfechos foi conduzida conforme o tempo para ocorrência do primeiro evento a partir do início da SCASSST pelo método de Kaplan-Meier com teste de log-rank para significância estatística entre as curvas de distribuição de sobrevida para os eventos . Os programas estatísticos utilizados foram o sistema R e o SPSS Statistics versão 19.0.Utilizamos os dados de mortalidade e IAM descritos na dissertação de mestrado (https://doi.org/10.11606/D.98.2020.tde-27122019-080250) de estudo conduzido na mesma UCO e estimamos uma diferença relativa de 50% na taxa de eventos entre os grupos com troponina negativa e troponina positiva. Adotando um poder de 90%, alfa de 5%, estimamos 273 casos como tamanho amostral mínimo para os objetivos, com testes de significância bilaterais. O nível de significância adotado foi bilateral < 0,05. As variáveis contínuas foram expressas em média e desvio padrão ou mediana com intervalo interquartil, conforme normalidade da distribuição das variáveis, a qual foi verificada pelo teste de Shapiro-Wilk. As análises de comparações entre os grupos foram efetuadas por oclearance de creatinina e de FEVE, e maior taxa de ocorrência de lesão renal aguda na fase hospitalar  .A mortalidade global foi 3,4%, sem diferenças entre os grupos, mas a incidência de IAM (ou reinfarto) foi 2-4 vezes maior no grupo de maior elevação de troponina . Curvas 30 Apesar do avanço terapêutico, há elevada morbimortalidade na fase precoce e após alta hospitalar, variando de 5-10% em 30 dias após o evento agudo até 20% em seis meses.31 Nesse contexto, diretrizes recomendam a troponina como biomarcador ideal para a estratificação de risco.33 Pacientes com maiores valores de troponina podem apresentar taxas de 20% de IAM e óbito em 30 dias, e de 25% em seis meses de evolução.35 Entretanto, diagnóstico diferencial de troponina elevada é essencial e deve ser analisada em conjunto com dados clínicos e exames complementares.28A DAC é uma das principais causas de óbito, principalmente quando analisamos o cenário clínico de SCA.13 Vale ressaltar a diferença considerável, em número absoluto, de IAM nos pacientes com troponina ≥0,12 ng/mL, mesmo em comparação àqueles com troponina >0,034 ng/mL.Neste estudo verificamos uma associação entre troponina minimamente elevada e maiores taxas de angiografia coronária e de procedimentos de revascularização. Isto pode ser justificado por esses pacientes apresentarem escores de risco mais elevados, o que aumenta a probabilidade de serem encaminhados para estratégia invasiva durante a hospitalização. Além disso, maiores taxas de IAM foram observadas entre os pacientes com níveis de troponina mais elevados. Este achado apresenta concordância com outros estudos nos quais foi demonstrada a associação do pico da troponina com maiores taxas de eventos adversos.10 Algumas observações podem explicar este achado. Primeiro, apesar do maior risco de óbito estimado pelo maior escore GRACE, elevação de troponina, gravidade anatômica coronária, e menor FEVE, a maioria dos pacientes dos três grupos foram encaminhados para estratificação invasiva. Assim, não houve associação linear isolada entre maior risco e maior nível de troponina, embora um maior número de pacientes com troponina elevada receberam tratamento de revascularização. Aliada aos fármacos de benefício comprovado na redução de eventos isquêmicos e óbito, essa abordagem mais “invasiva”, baseada não apenas em grandes elevações, mas também em pequenas elevações nos níveis de troponina, pode ter mitigado o desfecho composto que seria esperado pelo maior escore de risco na admissão hospitalar. Portanto, a estratégia invasiva foi relevante para a redução dos eventos cardiovasculares apesar do maior risco inicial. Isso é claramente corroborado pela redução da pontuação obtida no escore de risco GRACE entre a admissão e a alta hospitalar.Em termos de mortalidade, apesar de não detectarmos diferença estatisticamente significativa, houve maior número de óbitos nos grupos com troponina elevada, corroborando dados de estudos que evidenciam a relação entre o valor pico da troponina e mortalidade em SCA.stents ou maior número de vasos tratados cirurgicamente, o que salienta a plausibilidade biológica entre o pico da troponina e a complexidade anatômica coronária.9Outro dado relevante é a associação entre os dois grupos de troponina elevada, em comparação com pacientes com teste negativo quanto à revascularização miocárdica. De fato, maiores valores de troponina foram associados ao implante de múltiplos 25Por outro lado, embora a Definição Universal de IAM recomende a utilização do percentil 99 para diagnóstico de IAM, ainda há restrições à implementação desse critério pela variabilidade de kits e de valores de referência de troponina I entre os hospitais, podendo interferir na comparabilidade entre estudos clínicos e padronização de protocolos.Na comparação entre os grupos, embora o número de pacientes submetidos à angiografia coronária não ter sido diferente, a proporção foi significativamente maior conforme o nível de troponina detectado . Como salientamos acima, a elevada proporção de diagnóstico anatômico realizado por angiografia pode ter influenciado na decisão de proceder à revascularização miocárdica percutânea ou cirúrgica, mesmo no grupo com mínima elevação da troponina, sem significância estatística em relação à mortalidade, apesar da diferença numérica de sobrevida.Este estudo enfatiza a relevância da utilização do critério diagnóstico de IAM como proposto pela Definição Universal de IAM, especificamente em relação a três aspectos: predição de desfechos cardiovasculares maiores, indicação de estratégia invasiva e realização de procedimentos de revascularização miocárdica, ao compararmos pacientes com diferentes faixas de detecção de troponina I.14O racional da análise de pontos de corte de troponina ocorre devido à grande variabilidade entre hospitais com relação ao nível de troponina considerado para o diagnóstico de IAM, com cerca de 30% de laboratórios hospitalares seguindo as recomendações da Definição Universal de IAM.rule-in ou rule-out para IAM em sala de emergência, em casos de dor torácica, em pacientes com apresentação muito precoce. De modo oposto, porém complementar à utilidade clínica da troponina, avaliamos o papel prognóstico e a possível influência do ponto de corte na tomada de decisão quanto às estratégias de manejo de SCASSST em unidade de terapia intensiva e o potencial efeito de atenuação do risco precoce estimado por escores, mesmo em casos sem elevação de troponina, em comparação aos pacientes com “mínima” elevação, em decorrência de estratificação invasiva “agressiva” e revascularização apropriada. De fato, nossos dados reforçam a proposição da definição universal de IAM para utilizar o percentil 99 da troponina, minimizando liberações inadvertidas de casos considerados sem elevação da troponina compatível com IAM ao utilizar o ponto de maior acurácia em vez do percentil 99.Neste estudo, não avaliamos o uso da troponina de alta sensibilidade, pois o objetivo não foi a avaliação da utilidade do biomarcador em protocolos de stents coronários implantados (100% farmacológicos) e uso da artéria torácica interna esquerda, e volume expressivo de procedimentos de intervenção percutânea e revascularização cirúrgica. Tais aspectos poderiam explicar o baixo número de óbitos e IAM mesmo após procedimentos invasivos mais complexos, o que pode não ser aplicável a outros centros com características e infraestruturas diferentes. Ainda, esses fatores podem ter interferido na indicação de angiografia coronária na maioria dos pacientes, sem associação isolada e linear com os níveis de troponina e não necessariamente dependentes de escores de risco mais elevados. Portanto, a associação entre troponina e os desfechos, a estratificação de risco e a indicação de revascularização pode ser diferente em instituições sem disponibilidade de laboratórios de hemodinâmica ou cirurgia cardíaca. Em relação ao escopo do estudo, a grande variedade de kits de troponina I pode influenciar a tomada de decisão local e não ser concordante com nossos achados. Por fim, pela característica exploratória dos estudos observacionais, a variabilidade inerente à seleção de pacientes, e fatores de confusão não mensurados, salientamos que os resultados e conclusões devem ser considerados como indicativos, dando suporte à aplicabilidade clínica em coorte brasileira.O número de eventos observados pode ter reduzido o poder estatístico para detectar diferenças significantes em termos de mortalidade. Entretanto, o elevado índice de angiografia coronária, mesmo nos pacientes sem elevação ou com mínima elevação de troponina, e de subsequente revascularização coronária precoce pode ter reduzido o risco agudo estimado. Por isso, não se pode descartar a hipótese de que não tenha existido diferença na mortalidade pelo fato não ter sido ao acaso. Além disso, os dados foram derivados de único centro, refletindo a prática de uma instituição de ensino e pesquisa, com experiência histórica, alta disponibilidade de exames invasivos e não invasivos, indicadores de desempenho como prescrição de fármacos, Valores de troponina I acima do percentil 99 pela Definição Universal de IAM ou acima do ponto de corte de maior acurácia para o diagnóstico de IAM definido para o kit específico apresentam papel prognóstico em termos da ocorrência do desfecho composto de óbito e IAM até 30 dias da SCASSST. De modo mais relevante, níveis de troponina minimamente elevados agregam informação útil ao diagnóstico clínico e escores de risco na tomada de decisão, pela identificação de pacientes com maior probabilidade de benefício com estratificação invasiva e procedimentos de revascularização coronária, o que poderia explicar a atenuação do risco precoce de óbito associado à elevação desse biomarcador. 1 and important causes of disability, hospitalizations and death, mainly in low per capita income countries.3Cardiovascular diseases are the main cause of death among people older than 60 years in Brazil,4 In the context of acute myocardial ischemia, in addition to establishing the diagnosis of acute myocardial infarction (AMI), the measurement of troponin is useful for risk stratification in invasive strategies.5 Besides, peak troponin levels are correlated with the extension of necrosis and left ventricular ejection fraction (LVEF), which are important determinants of post-AMI mortality.7 In addition, elevated troponin levels have been correlated with multivessel coronary artery disease (CAD) and greater severity of stenosis in patients with non-ST-elevation acute coronary syndrome (NSTE-ACS),9 in addition to a directly proportional relationship with rates of clinical outcomes13 in patients undergoing early revascularization procedures.14In acute coronary syndromes (ACS), serial electrocardiography (ECG) and troponin measurements, associated with clinical examination, are essential for the correct diagnosis and appropriate management of disease.15 Among patients stabilized after an ACS, increased troponin is associated with higher all-cause cardiovascular mortality, regardless of covariables.16 Individuals with very high levels of troponin have more complex CAD and, based on pathophysiological plausibility, revascularization may be more often indicated in this group as compared with patients without troponin elevation. On the other hand, some authors have not found an association between high troponin levels and worse clinical outcomes.18Factors including severity and complexity of CAD, previous use of acetylsalicylic acid and early coronary angiography are associated with peak troponin in NSTE-ACS.th percentile of a healthy population, and an intra-assay coefficient of variation (CV) <10%,19 although many troponin test kits have poor accuracy based on this percentile.22 Therefore, for the use of troponin in the diagnosis of AMI, it is necessary an ascending or descending curve of the biomarker, including at least one value above the 99th percentile of the reference population, according to sex, ethnics and other factors.24 Some studies have pointed out the importance of using standard levels for the diagnosis of AMI in hospital laboratories to improve clinical decisions, to tailor diagnostic thresholds to the population seen in each institution, and facilitate reporting in clinical trials.26According to the International Federation of Clinical Chemistry and the National Academy of Clinical Biochemistry, increased troponin levels are defined as those above the 99th percentile), mild increase , and significant increase  in patients at the early stage of NSTE-ACS; to compare three groups formed according to the ranges of troponin I values – without elevation , divided into groups according to troponin levels. All clinical events were pre-defined and assessed following a systematic collection of data from databases. Indication for invasive or non-invasive risk stratification, in-hospital treatments, and routine laboratory tests were also evaluated. All participants signed an informed consent form. The study was approved by local ethics committee in April 2019. Recruitment of patients was carried out from May 2019 to January 2020.•  Age ≥ 18 years•  Admission to a CCU•  Diagnosis of NSTE-ACS•  The diagnosis of NSTE-ACS was made based on two of the following criteria:- Clinical presentation suggestive of ACS;- ECG showing depression of the ST segment, T-wave inversion or non-specific findings;- Ascending or descending troponin curves, including at least one value above 0.12 ng/mL .Reinfarction was defined following the recommendations of the fourth universal definition of AMI, and considered suspected in the presence of signs or symptoms of infarction, requiring another troponin measurement in this case. Diagnosis is confirmed by a 20% increase in troponin levels in patients with already elevated values, or a new increase in those with previously normal levels.•  The diagnosis of unstable angina was made based on two of the following criteria:- Clinical presentation suggestive of ACS;- ECG showing depression of the ST segment, T-wave inversion or non-specific findings;27 and the European Society of Cardiology guidelines28).- Absence of troponin levels above 0.034 ng/mL , GRACE and CRUSADE scores were analyzed. Laboratory tests, therapeutic procedures and approaches during hospital stay were performed according to institutional protocols.th percentile value of 0.034 ng/mL, diagnostic cut off point of 0.12 ng/mL for AMI, sensitivity of 95% and specificity of 93%  was used for measurements of cardiac troponin I, with a 99y of 93% . The CV We used the mortality and AMI data described on a Masters thesis of a study conducted at the same CCU (available at: https://doi.org/10.11606/D.98.2020.tde-27122019-080250), and estimated a relative difference of 50% in the rate of events between the groups with negative and positive troponin. Using a power of 90% and alpha of 5%, we estimated a minimum sample size of 273 patients for the objectives of the study. Two-sided significance tests were used, with significance level at 0.05. Continuous variables were expressed as mean and standard deviation or median and interquartile range, according to normality of distributions, which was tested using the Shapiro-Wilk test. Between-group comparisons were assessed by the one-way ANOVA or by the non-parametric Kruskal-Wallis test. Categorical variables were expressed as frequency and percentages and compared by the chi-square test or Fisher exact test. Analysis of outcomes was conducted according to the time to the first event since the onset of NSTE-ACS by the Kaplan-Meier method and the log-rank test for statistical significance between survival curves for the events . Statistical analysis was performed using the R system and the SPSS statistics, version 19.0.A total of 494 patients with diagnosis of NSTE-ACS were evaluated. A greater number of patients with significant increase in troponin levels underwent functional studies, invasive approach, and myocardial revascularization procedures. All patients were treated with benefit-proven medications recommended by guidelines  .Overall mortality was 3.4%, with no statistically significant difference between the groups, although the incidence of AMI (or reinfarction) was 2-4 times higher in the group with troponin elevation . Kaplan–30 Despite therapeutic advances, morbidity and mortality of CAD in the early stages of disease and after hospital discharge are high, varying from 5-10% within 30 days to 20% in six months after the acute event.31 In this context, guidelines have recommended the use of troponin as a biomarker for risk stratification.33 Patients with increased troponin may have a 20% rate of AMI and death in 30 days, and a 25% rate in six months of follow-up.35 However, differential diagnosis by increased troponin is crucial and should be analyzed together with clinical data and complementary tests.28CAD is one of the main causes of death, especially in the context of ACS.13 It is worth mentioning the considerably higher number of cases of AMI among patients with troponin levels ≥0.12 ng/mL, even as compared with those with troponin > 0.034 ng/mL.In the present study, we observed an association between mild elevations of troponin and higher rates of coronary angiography and revascularization procedures. This may be explained by the fact that these patients had higher risk scores, which increases the likelihood of referral for invasive procedures during hospitalization. Also, higher rates of AMI were found in patients with increased troponin. This is in agreement with previous studies showing that peak troponin values are associated with higher rates of adverse events.10 These data may be explained by some factors. First, despite the higher risk of death estimated by higher GRACE score, troponin elevation, severity of CAD, and lower LVEF, most patients of the three groups were referred for invasive risk stratification. Therefore, there was not a linear association between increased risk and elevated troponin, although a larger number of patients with a marked increase in troponin levels underwent revascularization. Combined with the use of medications with proven benefits in reducing ischemic events and death, this “more invasive” approach, based not only on significant elevations but also on mild elevations in troponin levels, may have mitigated the occurrence of composite events that would be expected due to high risk score at admission. Therefore, the invasive strategy was important to reduce cardiovascular events despite increased initial risk. This is clearly corroborated by the reduction in GRACE score from hospital admission to discharge.Regarding mortality rates, although we have not detected statistically significant difference, a higher number of deaths was found among patients with elevated troponin, corroborating other studies that showed a relationship between peak troponin values and mortality in ACS.9Another important finding is the association between the two groups of patients with elevated troponin in comparison with patients with normal levels regarding myocardial revascularization. Higher troponin levels were associated with multiple stent implantation and higher number of surgically treated vessels, which reinforces the biological plausibility linking increased troponin levels with complexity of coronary anatomy.th percentile for the diagnosis of AMI, its widespread implementation is still hampered by between-kit variability and variation of troponin I reference values between hospitals, which may influence the comparability between clinical trials and standardization of protocols.25On the other hand, although the universal definition of AMI recommends the use of the 99In the comparison between the groups, although the number of patients who underwent coronary angiography was not different, the proportion of patients was significantly higher in those with greater elevation of troponin . As we pointed out, the high proportion of anatomic diagnosis made by angiography may have influenced the decision to perform percutaneous or surgical myocardial revascularization, even in patients with mild increase of troponin, with no statistical difference regarding mortality, despite numerical difference in survival.This study emphasizes the importance of using the diagnostic criteria of AMI proposed by the universal definition of AMI, particularly with respect to three aspects – prediction of major cardiovascular outcomes, indication of invasive strategy, and performance of myocardial revascularization procedures in the comparison of groups of patients with different troponin levels.14The rationale of the analysis of troponin cutoff points is the high variability of the diagnostic value between the hospitals, with nearly 30% of hospital laboratories following the recommendations by the universal definition of AMI.th percentile concentration of cardiac troponin proposed by the universal definition of AMI, avoiding unnecessary dismissal of patients without troponin elevations compatible with AMI when adopting a more accurate cutoff instead of the 99th percentile.In the present study, we did not evaluate the use of high-sensitivity troponin since our objective was not to analyze the usefulness of this biomarker in rule-in or rule-out protocols for AMI in the emergency room in cases of chest pain, in patients with very early presentation. However, we did analyze the prognostic role and the possible influence of troponin cutoff values on the decision making in the management of NSTE-ACS in intensive care unit as well as the potential effect of attenuating the early risk (estimated by scores), even in patients without troponin elevation, as compared with patients with slight elevation, by an “aggressive” invasive risk stratification and appropriate revascularization. Indeed, our data reinforce the use of the 99The number of events observed may have reduced the statistical power to detect significant differences in terms of mortality. However, the high rate of coronary angiography, even among patients with no or minimal elevation of troponin, and subsequent early coronary revascularization may have reduced the estimated acute risk. Therefore, the hypothesis of a difference in mortality cannot be excluded, as this was not a random situation. Second, our data derived from a single center and reflected the reality of a research and education institution, with historical experience, and where invasive and non-invasive tests, indicators of performance such as drug prescription, coronary stent implantation (100% drug-eluting stents) with use of the left internal thoracic artery are highly available, and a large volume of percutaneous intervention and surgical revascularization has been performed. These factors could probably explain the low number of deaths and AMI even after highly complex invasive procedures, which may not be applied to other centers with different characteristics and infrastructures. Also, these aspects may have influenced the indication for coronary angiography in the majority of patients, without an isolated and linear association with troponin levels, and not necessarily dependent on more elevated risk scores. Thus, the association of troponin with outcomes, risk stratification and indication of revascularization may be different in institutions where hemodynamic laboratories and cardiac surgery are not available. Considering the scope of the study, the high variability of troponin I kits may influence local decisions and produce divergent results. Finally, due to the exploratory nature of observational studies, variability inherent to the selection of patients, and unmeasured confounding factors, we emphasize that the results and conclusions obtained in this study should be considered just an indication and be used as a support to their applicability in Brazilian populations.th percentile, proposed by the universal definition of AMI, or above the most accurate diagnostic cut-off point for AMI, defined by specific kit, have prognostic value in terms of the occurrence of composite endpoint of death and AMI within 30 days after NSTE-ACS. More importantly, mild elevations of troponin add useful information to the clinical diagnosis and risk scores in the decision-making process, by identifying those patients who would most benefit from invasive risk stratification and coronary revascularization procedures, which could explain the attenuation of early mortality risk associated with the increase in this biomarker.Cardiac troponin values above the 99


Whereas significant strides have been made in the treatment of lysosomal storage diseases (LSDs), the neuronopathy associated with these diseases remains impervious mainly because of the blood-brain barrier (BBB), which prevents delivery of large molecules to the brain. However, 100 years of research on the BBB since its conceptualization have clarified many of its functional and structural characteristics, spurring recent endeavors to deliver therapeutics across it to treat central nervous system (CNS) disorders, including neuronopathic LSDs. Along with the BBB, the cerebrospinal fluid (CSF) also functions to protect the microenvironment of the CNS, and it is therefore deeply involved in CNS disorders at large. Recent research aimed at developing therapeutics for neuronopathic LSDs has uncovered a number of critical roles played by the CSF that require further clarification. This review summarizes the most up-to-date understanding of the BBB and the CSF acquired during the development of therapeutics for neuronopathic LSDs, and highlights some of the associated challenges that require further research. Lysosomal storage diseases (LSDs) are progressive inherited disorders of the metabolism, in which, due to a genetic deficiency of lysosomal enzymes, substrates remain unmetabolized and accumulate inside the organelles of the endosomal-autophagic-lysosomal system. About two-thirds of patients with LSDs exhibit complex central nervous system (CNS) dysfunctions. Such neuronopathic LSDs include mucopolysaccharidoses , sphingolipidoses, mucolipidoses, oligosaccharidoses, multiple sulfatase deficiency, and neuronal ceroid lipofuscinoses Sun .The recent advent of novel treatments for LSDs  has benefited patients by alleviating their symptoms and improving prognoses  both play critical roles in preserving the intricate structures and functions of the brain by protecting it from external insults, and from pathogenic and toxic substances Proulx . UnfortuHowever, better understanding and therapeutic utilization of the BBB and the CSF are of paramount importance for the development of novel therapeutics to address CNS disorders in patients with neuronopathic LSDs. A wide range of issues need to be addressed, from clarifying the underlying pathogenesis of neurodegeneration, developing and manufacturing BBB-crossing enzymes, to evaluating the optimal drug effects against CNS symptoms in clinical trials. Recent research into the BBB and CSF has been delineating the complex physiological nature of these fundamental mechanisms, which are still far from being fully elucidated. This review attempts to summarize recent advances in our understanding of the BBB and the CSF which need to be taken into account during research aimed at developing effective treatments for neuronopathic LSDs.General structures and functionsOriginally conceptualized by Lewandowsky  followinDysfunction of the BBB is known to play a central role in a number of neurodegenerative diseases (e.g. Alzheimer’s disease and multiple sclerosis), whereby the disrupted BBB causes ion dysregulation and allows neurotoxic blood-derived debris, cells, and microbial pathogens to enter the brain; this leads to inflammation, opening up multiple possible pathways towards neurodegeneration Transcytosis through the BBB – intracellular transport in cerebrovascular endothelial cells –Table Perhaps due to the specialized tight junctions that prevent paracellular passage between the blood and the brain, transcellular pathways have been a major focus of attention as a means of drug delivery. In particular, transcytosis  is considered critical in overcoming the restrictive properties of the BBB Drug delivery to the brain via receptor-mediated transcytosisTranscytosis is initiated by endocytosis, a process by which substances in the luminal spaces are internalized by endothelial cells, and the internalization process can proceed via three pathways: the caveolae, clathrin-dependent endocytosis, or clathrin-independent endocytosis  for neuronopathic LSDs. Five compounds that harness receptor-medicated transcytosis have so far progressed to clinical trials Table .Table 2CAs an example, a schematic illustration showing the mechanism of transcytosis via transferrin receptors is given in Fig. Transferrin binds to the transferrin receptors located on the luminal side of the microvascular endothelial cells in the brain and is absorbed into the endothelial cells (endocytosis), in which it is then transported towards the abluminal side of the cell facing the brain parenchyma, and subsequently released from the receptors to reach the brain parenchyma (exocytosis). Likewise, enzymes fused with an anti-transferrin receptor antibody bind to the transferrin receptors, are absorbed into the endothelial cells, and are then released into the abluminal side of the cells so that they can diffuse into the brain parenchyma.There have been a number of translational challenges in establishing drug delivery via receptor-mediated transcytosis. One of them is that using receptor-mediated transcytosis of targeted enzymes can affect the original innate transcytosis that is supposed to deliver physiological substances like transferrin , α-L-iduronidase fused with anti-insulin receptor antibody, in patients with MPS-I detected drug-related transient hypoglycaemia in 6.4% of the patients , iduronate-2-sulfatase fused with anti-transferrin receptor antibody, on patients with MPS-II, there were no adverse events associated with the iron metabolism, which it was thought the drug might affect -containing glycans on the lysosomal enzyme that mediate enzyme uptake by the M6P receptor are present on most cells in the body; hence the resultant somatic efficacy of the fusion protein. The affinity of the M6P-containing glycans to the M6P receptor is in the low nM range, ensuring rapid cellular uptake of the enzyme  active CSF formation (secretion), 2) passive CSF absorption (drainage), and 3) unidirectional CSF flow from its formation sites to absorption sites  that is almost antithetical to the traditional view has recently been put forth  that remain uncatabolized because of the enzyme deficiency seen in patients with neuronopathic LSDs are by far the most important determinant in initiating the complex cascade of neurodegenerative processes (Sato and Okuyama In an established animal model of MPS II, close correlations were observed between intracerebral and CSF HS levels, and intravenous administration of pabinafusp alfa led to dose-dependent reductions in HS levels in the CSF, along with corresponding reductions in HS levels in the brain (Sonoda et al. IT administration of enzymes in patients with MPS-II (Muenzer et al. The blue arrows represent the innate natural circulation of the CSF, the green arrows intravenously administered BBB-crossing enzymes, the black arrows intrathecally administered conventional enzymes, and the yellow ones intracerebroventricularly administered conventional enzymes (Modified from Tumani et al. IT administration of enzymes can, by definition, directly affect the substrate levels in the subarachnoid space, while their penetration into the brain parenchyma to act against CNS disorders is very limited, as discussed above. In contrast, BBB-crossing enzymes administered intravenously can traverse the BBB via receptor-mediated transcytosis, thus addressing the brain parenchyma widely and directly, where they reduce the substrate accumulations therein. The enzymes are thought to be mostly consumed in the process, so that few, if any, remnants circulate into the CSF. Therefore, changes in CSF substrate levels brought about by intravenous ERT with BBB-crossing therapeutics truly reflect the changes in intracerebral substrate levels.sui generis of the CSF and BBB have to be taken into account when evaluating the pharmacokinetics and pharmacodynamics of drugs targeting the CNS.As shown in Fig. The year 2021 marks the centenary of the naming of the BBB by Lina Stern (1878–1951; Dreifuss and Tikhonov However, there are still a number of remaining issues surrounding the emerging ERTs for neuronopathic LSDs. First, details of the specific regulating processes in various modes of transcytoses need to be clarified so that drug delivery via transcytosis can be optimized to consolidate efficiency, efficacy, and safety. Second, the CSF microcirculation has only been partly elucidated, and its pharmacokinetic and pharmacodynamic relevance to ERT and other treatment modalities for neuronopathic LSDs needs further investigation. Third, knowledge about how the neuronopathy seen in patients with LSDs responds to treatment is still scant, which means that valid and reliable methods of evaluating the efficacy of novel therapeutics are yet to be established. In other words, with the advent of new therapies to address the neurological and psychiatric signs and symptoms of patients with neuronopathic LSDs, we need to learn to understand how these new treatments influence an affected individual in the way he/she feels, functions, and survives. Also, the inevitable paucity of patient populations and significant heterogeneity in the clinical manifestations and prognoses of these rare diseases impede the efficient conduct of clinical trials and cause further delays in introducing novel treatments. Last but not least, designing and manufacturing a fusion protein for efficient transcytosis-mediated drug delivery entails formidable challenges in establishing acceptable pharmacokinetic and pharmacodynamic profiles, stability, yield, and other production-related technical specifications.It is hoped that the wide-ranging research being carried out will lead to further insights into neuronopathic LSDs, facilitating the development of effective treatments for patients suffering from these rare, debilitating diseases.


Parvoviridae comprises many major viral pathogens that can infect humans and multiple other species, causing severe diseases. However, knowledge of parvoviruses that infect equids is limited. In the present study, we found that three equine parvoviruses (EqPVs), namely, equine parvovirus-hepatitis (EqPV-H), equine parvovirus-cerebrospinal fluid (EqPV-CSF) and equine copivirus (EqCoPV) cocirculated among horses in China. We examined the prevalence of these three EqPVs in 225 horse serum samples in China and found EqPV-H, EqPV-CSF and EqCoPV viremia in 7.6% (17/225), 2.7% (6/225) and 2.2% of samples (5/225), respectively. We also obtained the complete genomes of one EqPV-H strain, six EqPV-CSF strains and one EqCoPV strain. After phylogenetic analysis of the EqPVs, we found that EqPV-CSF and EqCoPV may have evolved from the same ancestor. The EqPV-CSF strains (E111 and A27) and EqCoPV strain (F124) were genetically similar to foreign strains, but the EqPV-CSF strains  comprised unique clades. This study determined the prevalence of three EqPVs in Chinese horses and analyzed the genetic characteristics of EqPVs prevalent strains in Chinese horse herds. Our data provide a theoretical basis for follow-up research on the prevention and control of EqPVs.The family  Parvoviridae is a diverse family of nonenveloped and small single-stranded DNA viruses. It is divided into three subfamilies, Densovirinae, Hamaparvovirinae and Parvovirinae, which contain 11, 5 and 10 genera, respectively . The members of Parvovirinae can infect multiple species, causing severe diseases, as established in the early twentieth century  was first reported in 1985 in Canada; it was discovered in the liver and blood of an aborted equine fetus . Subsequ in 2015 . In 2018 in 2015 . In 2019 in 2015 .Parvoviridae, is a small, nonenveloped DNA virus. Its genome has two open reading frames encoding nonstructural protein 1 (NS1) with a length of 566–594 amino acids and virion protein 1 (VP1) with a length of 975–1,078 amino acids. The pathogenicity of EqPVs remains unclear. However, despite serving as a potential pathogen of equine hepatitis, EqPVs have not attracted the attention of Chinese veterinarians  assays to detect EqPV DNA. Detailed genetic and clinical analyses were conducted.A total of 225 equine serum samples was collected from seven racetracks in Guangdong Province in 2019-2020, and the characteristics  of each horse were recorded. None of the horses that the samples were collected from were clinically apparent. All 200 μL samples were aliquoted into 1.5 mL EP tubes and stored at −80°C.Escherichia coli (E. coli) DH5α-competent cells . After culturing for 10 h, bacterial clones were identified by PCR, and positive E. coli clones were identified; the positive samples were sent out for direct Sanger sequencing . BLAST analysis of the sequencing results of positive samples was conducted in the National Center for Biotechnology Information (NCBI) database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).DNA from serum samples was extracted from 200 μL of the supernatant using a MiniBEST Viral RNA/DNA Extraction Kit  according to the manufacturer's instructions and then stored at −80°C until further use. To detect the presence of EqPVs, all samples underwent nested PCR using specific primers that targeted the NS1 gene. The primers EqPV-H-F1 and EqPV-H-R1 were used in the first round of PCR and EqPV-H-F2 and EqPV-H-R2 were used in the second round of PCR to detect EqPV-H; the primers EqPV-CSF-F and EqPV-CSF-R were used to detect EqPV-CSF; and the primers EqCoPV-F and EqCoPV-R were used to detect EqCoPV . The tarE. coli DH5α-competent cells . After culturing for 10 h, bacterial clones were identified by PCR, and the positive E. coli clones were identified and sent for sequencing . BLAST analysis of the sequencing results of positive samples was conducted in the NCBI database. Then, virus nucleotide sequences were assembled with SeqMan7.1.0.The genome sequences of all EqPV-CSF, EqPV-H, and EqCoPV strains were downloaded from the NCBI database and compared in BioEdit 7.0.9.0. Multiple pairs of primers for the conserved region were designed to cover the complete genomes of EqPV-CSF, EqPV-H, and EqCoPV in Oligo 7.0 . The tarCopiparvovirus and all EqPV-CSF, EqPV-H, and EqCoPV strains.The nucleotide sequences of the positive samples were aligned using BioEdit 7.0.9.0, and the nucleotide identities were derived. Phylogenetic trees were established with MEGA 6.0 using the maximum likelihood method with the JTT+G model based on the bootstrap values of 1,000 replicates. A phylogenetic tree was constructed based on the published NS1 amino acid sequences of In this study, we collected 225 equine serum samples from seven farms in Guangdong Province, including an equestrian training center . To inveAfter PCR and agarose gel electrophoresis analysis, a total of 24 equine serum samples presented a band with a size similar to the expected size of the EqPVs. Sequencing and online BLAST analysis showed that 17 samples were positive for EqPV-H DNA (7.6%), 6 samples were positive for EqPV-CSF (2.7%) and 5 samples were positive for EqCoPV (2.2%). These results demonstrated that three novel parvoviruses  circulated in equine populations at these seven farms. However, all EqPV-positive horses were clinically healthy.Detailed information on the equine samples positive for EqPV DNA is provided in OM31076-OM310771 and OM310773).To date, the whole genomes of a total of 16 EqPV-H strains, 3 EqPV-CSF strains and 4 EqCoPV strains have been reported. To better understand the genetic characterization of the three common EqPVs in China, we amplified the nearly complete viral genome of EqPVs with gap-filling PCR . All genThe EqPV-H genome is 5,308 nucleotides long and contains NS1 , intergenic region 1, VP1  and intergenic region 2. After sequence alignment and analysis of American, Korean, and Chinese strains, we found nucleotide identities of 95.8–99.9% and 95.2–100% for NS1 and VP1, respectively, among EqPV-H strains . Amino aEqPV-CSF is a newly discovered EqPV; thus, genome sequences of its strains are relatively scarce, and only 3 strains in the NCBI database have relatively complete genomes. In this study, we successfully obtained nearly complete genomes (missing the first 155 nucleotides of the NS1 gene) of 6 EqPV-CSF strains with PCR. After sequence alignment and analysis, we found that the GC contents of the six EqPV-CSF strains were in the range of 42.30–42.71%. When these strains were compared with previously reported EqPV-CSF strains in the USA and South Korea, no nucleotide insertions or deletions were found. At the nucleotide level, six EqPV-CSF strains had similarities of 92.9–99.6% and 92.2–98.9% in the NS1 and VP1 genes, respectively. Compared to other EqPV-CSF strains, these six strains had similarities of 83.2–99.5% and 91.9–95.8% of the NS1 and VP1 genes, respectively . At the Similar to EqPV-CSF, sequence information for EqCoPV in the NCBI database is relatively scarce; there are only five complete genome sequences of EqCoPV. Our study is the first to obtain complete NS1 and VP1 sequences for the EqCoPV strain in China . After sCopiparvovirus , 2.7% (6/225) and 2.2% (5/225), respectively, as determined with PCR. The prevalence of EqPV-H was similar to that previously reported 11.9%), while the prevalence of EqPV-CSF was lower than that previously reported in Xinjiang (25%) , 16. Pre%, while We found horses that were coinfected with EqPV-H and EqPV-CSF or with EqPV-H and EqCoPV but no horses coinfected with EqCoPV and EqPV-CSF or with three EqPVs. Interestingly, this result is similar to our previous study on EqPV-H, equine hepacivirus (EqHV), equine pegivirus (EPgV), and Theiler's disease-associated virus (TDAV), which found coinfection of EqPV-H and other blood-borne viruses . FurtherIn the phylogenetic tree analyses, the Chinese EqPV-CSF and EqCoPV strains were highly similar to strains isolated from the USA and South Korea, while EqPV-H was placed in its own clade. This finding indicates that both EqPV-CSF and EqCoPV evolved from a common ancestor. More importantly, we found that not only were the Chinese EqPV-CSF E111 and A27) strains and an EqCoPV (F124) strain genetically similar to foreign strains but the EqPV-CSF strains  were comprised of unique clades. EqPV-CSF was reported in China in 2018; the report presented valuable information showing that EqPV-CSF-like virus was likely exotic and introduced into Xinjiang through international horse trade. However, through our phylogenetic analysis, we found that the EqPV-CSF  strains were divided into independent clades. This suggests that EqPV-CSF may have been circulating in China prior to 2018 and evolved into a separate branch. Previous studies have found that there is a phenomenon of strain recombination in EqPV-H  can be found in the article/No studies involving human participants or animals performed by any of the authors are described in this article. This study uses 225 samples obtained from racetracks in Guangdong province. The South China Agricultural University Experimental Animal Welfare Ethics Committee did not require the study to be reviewed or approved by an ethics committee because all the samples in this study originate from diagnostic samples. Written informed consent was obtained from the owners for the participation of their animals in this study.JO performed the experiments and analyzed the data. XW, JL, JX, GL, and SL participated in whole study design, coordinated the study, and prepared the manuscript and manuscript revision. LX and LZ assisted with conducting the experiments. All authors have read and agreed to the published version of the manuscript.


A troponina agora também pode ser detectada em cenários atípicos, como em exercícios de resistência extenuante, estimulação atrial rápida e ecocardiografia de estresse com dobutamina, bem como em 50% a 100% dos indivíduos saudáveis.Atualmente, os ensaios de troponina cardíaca de alta sensibilidade (hs-cTn) estão disponíveis para uso clínico e fazem parte da definição de infarto agudo do miocárdio (IAM).fast twitch), esquelética de contração lenta (slow twitch) e cardíaca-específica . Por outro lado, a TnC tem duas isoformas, uma presente no músculo esquelético de contração rápida (fsTnC) e outra com expressão tanto no músculo esquelético de contração lenta quanto no músculo cardíaco (ssTnC /cTnC).Do ponto de vista fisiológico, a troponina é um complexo proteico que regula a função miofibrilar, formado por três subunidades: I, T e C. No caso da troponina I e da troponina T, existem 3 isoformas tecido-específicas diferentes: troponina esquelética de contração rápida . Essa ferramenta é aplicada para prever se uma determinada proteína sofre secreção sem sinalização de peptídeo. É um método baseado em sequência que, com a ajuda de redes neurais, detecta características específicas comuns a proteínas extracelulares/secretadas.Para avaliar a secreção não clássica de troponina, foi utilizado o servidorSignalP 5.0, um método comumente utilizado para o reconhecimento de sinalização de peptídeos com base em redes neurais . Esta etapa inicial foi realizada para excluir a via secretora clássica. Em seguida, as sequências foram analisadas comSecretomeP 2.0. Finalmente, para avaliar a possibilidade de secreção não clássica do tipo IV (utilizada por proteínas transmembranares que se desviam do Complexo de Golgi), as sequências foram avaliadas utilizando oTMHMM 2.0, um programa projetado para detectar hélices transmembranares (http://www.cbs.dtu.dk/services/TMHMM/). Uma representação gráfica do pipeline de bioinformática que foi seguido é mostrada na Figura Suplementar 1.As sequências canônicas das três subunidades de troponina do músculo esquelético de contração rápida, contração lenta e músculo cardíaco foram obtidas do banco de dados UniProtKB (Arquivo Suplementar 1). As proteínas foram primeiramente examinadas comCerca de 19.900 artigos foram revisados, dos quais 31 foram lidos na íntegra. Após a avaliação do resumo e do texto completo, não foram encontrados artigos relacionados à secreção de troponina. Portanto, que seja de nosso conhecimento, este é o primeiro artigo relatando evidências de secreção não clássica de troponina.SecretomeP 2.0 previu que cTnT, fsTnI, ssTnI, fsTnT e fsTnC eram secretadas de forma não clássica. Nos 5 casos, as proteínas alcançaram um escore de rede neural (escore NN) superior a 0,6, que é o limite mínimo para sequências em mamíferos. Além disso, nenhuma das oito isoformas da troponina mostrou conter uma sinalização de peptídeo ou uma hélice transmembranar, de acordo comSignalP 5.0eTMHMM 2.0, respectivamente. Os resultados resumidos são apresentados naApós analisar as isoformas da troponina, o Consequentemente, ela não necessita de uma sinalização de peptídeo, que é uma sequência curta de aminoácidos que conduz a proteína através do processo secretório clássico (mediado pelo RE/Golgi). Em vez disso, as proteínas secretadas de forma não clássica são liberadas através de uma miríade de mecanismos que podem ser classificados em 4 grupos: tipo I (transporte dependente de poros), tipo II (liberação mediada por transportador ABC), tipo III (liberação através de endossomos/autofagossomos) e tipo IV (desvio do Complexo de Golgi por proteínas transmembranares). Além disso, outros mecanismos, como exossomos e bolhas, também foram reconhecidos como participantes da secreção não clássica. Curiosamente, a maioria dos cenários de secreção não-convencional são desencadeados por estresse celular, como inflamação. Alguns exemplos de proteínas que utilizam a via não clássica são IL-1β/IL-1α, FGF-1, FGF-2 e galectinas.A secreção não clássica ou não convencional é uma via de liberação de proteínas. Ao contrário da secreção clássica, ela é independente do retículo endoplasmático (RE)/Complexo de Golgi. Não é irracional pensar que uma condição de atividade geradora de estresse no célula cardíaca resulta na liberação de troponina através de um processo secretor não clássico  já foram demonstradas anteriormente. As evidências disponíveis estão resumidas naPesquisas futuras devem ter como objetivo demonstrar a troponina circulante dentro das microvesículas. Isso pode ser testado em pacientes com infarto agudo do miocárdio  versus cenários atípicos apresentando elevação da troponina, como exercício extenuante, sepse e acidente vascular cerebral (onde a secreção não clássica deve conduzir a liberação de troponina). Após o isolamento das microvesículas no plasma, uma possível abordagem para validar a via de secreção não clássica poderia ser o uso de espectrometria de massas seguida por confirmação através de anticorpos específicos.in silico, ela deve ser confirmada experimentalmente antes que seu significado clínico exato possa ser determinado. Se outras variantes da troponina também entram na via secretora não clássica permanece uma questão em aberto. No entanto, é justo dizer que a secreção não clássica da troponina é uma linha de pesquisa promissora em cardiologia que aguarda ser explorada.Ensaios de alta sensibilidade trouxeram incertezas consideráveis sobre as elevações de cTn. A liberação de troponina devido a causas não relacionadas ao IAM permanece indefinida e seu significado prognóstico exato continua a ser investigado. Como nossa proposta é baseada em evidênciasPara informação adicional, por favor,clique aqui “It is important to realize that if certain areas of scienceappear to be quite mature, others are in the process ofdevelopment, and yet others remain to be born.”Santiago Ramón y Cajal,Advice for a young investigatorHowever, troponin elevation is not limited to AMI, since other conditions related to oxygen demand mismatch, direct myocardial damage, increased myocardial strain, systemic processes (i.e. sepsis), neurological disease and renal failure can also result in its increase.Troponin can now also be detected in atypical scenarios, such as in strenuous endurance exercise, rapid atrial pacing, and dobutamine stress echocardiography, as well as in 50% to 100% of healthy subjects.Nowadays, high-sensitivity cardiac troponin (hs-cTn) assays are available for clinical use and are part of the definition of acute myocardial infarction (AMI).Conversely, TnC has two isoforms, one present in fast-twitch skeletal muscle (fsTnC) and one that expresses in both slow-twitch skeletal and cardiac muscle (ssTnC/cTnC).From a physiological point of view, troponin is a protein complex that regulates myofibrillar function, consisting of three subunits: I, T and C. In the case of troponin I and troponin T, there are three different tissue-specific isoforms: fast-twitch skeletal, slow-twitch skeletal and cardiac specific .However, none of them concisely explains why troponin is released in cases unrelated to myocardial infarction or whether this is indicative of reversible or permanent damage to the cardiac cell. In light of the current lack of knowledge, our main objective was to explore the feasibility of a novel mechanism for cardiac troponin release using a bioinformatical approach.Apart from cell necrosis, several alternative release mechanisms have been postulated for the cardiac isoforms of troponin.Before starting the analysis, a comprehensive review of the literature was conducted in search of articles related to troponin secretion. The searched databases were PubMed, bioRxiv and OpenGrey. Articles were first assessed based on their title. If it mentioned troponin and a secretory process, the abstract was read. Articles were selected if the abstract mentioned troponin and a secretory process or pathway. Purely clinical articles that did not investigate a mechanism of troponin release were excluded. The screening and review processes were performed by all authors. Disagreements were solved by consensus during regular meetings.SecretomeP 2.0server was used (http://www.cbs.dtu.dk/services/SecretomeP/).This tool is applied to predict whether a certain protein undergoes secretion without a signal peptide. It is a sequence-based method that, with the help of neural networks, detects specific features common to extracellular/secreted proteins.To assess for non-classical secretion of troponin, theSignalP 5.0, a commonly used method for the recognition of signal peptides based on neural networks .This initial step was performed to rule out the classical secretory pathway. Afterwards, the sequences were analyzed withSecretomeP 2.0. Finally, to assess the possibility of type IV non-classical secretion (used by transmembrane proteins that bypass the Golgi apparatus), the sequences were evaluated usingTMHMM 2.0, a program designed to detect transmembrane helices (http://www.cbs.dtu.dk/services/TMHMM/).A graphical representation of the followed bioinformatic pipeline can be found in SupplementaryThe canonical sequences from the three subunits of troponin from fast-skeletal, slow-skeletal and cardiac muscle were retrieved from the UniProtKB database (Supplementary File 1). The proteins were first assessed withNearly 19,900 articles were reviewed, of which 31 were read in full. After abstract and full text assessment, no articles were found to be related to troponin secretion. Hence, to the best of our knowledge, this is the first paper reporting evidence of troponin non-classical secretion.SecretomeP 2.0predicted cTnT, fsTnI, ssTnI, fsTnT and fsTnC to be non-classically secreted. In the 5 cases, the proteins achieved a neural network score (NN-score) greater than 0.6, which is the minimum threshold for mammalian sequences. Additionally, none of the eight troponin isoforms were found to contain a signal peptide or a transmembrane helix, according toSignalP 5.0andTMHMM 2.0, respectively. The summarized results are shown inAfter analyzing the troponin isoforms,Consequently, it does not require a signal peptide, which is a short sequence of amino acids that leads the protein through the classical (ER/Golgi-mediated) secretory process.Instead, non-classically secreted proteins are released through a myriad of mechanisms that can be classified into 4 groups: type I (pore-dependent transport), type II (ABC transporter-mediated release), type III (released from endosomes/autophagosomes) and type IV (Golgi bypass by transmembrane proteins).Moreover, other mechanisms, such as exosomes and blebs, have also been recognized to take part in non-classical secretion.Interestingly, most of the scenarios of unconventional secretion are triggered by cell stress, such as inflammation. Some examples of proteins that use the non-classical pathway are IL-1β/IL-1α, FGF-1, FGF-2, and galectins.Non-classical or unconventional secretion is a pathway of protein release. Contrary to classical secretion, it is independent of the Endoplasmic Reticulum (ER)/Golgi apparatus.It is not unreasonable to think that an activity condition that generates stress in the cardiac cell results in the release of troponin through a non-classical secretory process  have been previously demonstrated. Available evidence is summarized inFurther research should aim at demonstrating circulating troponin within microvesicles. This could be tested in patients with acute myocardial infarction (where necrosis should be the leading release mechanism) versus atypical scenarios showing troponin elevation such as strenuous exercise, sepsis and stroke . After the isolation of plasma microvesicles, a possible approach to validate the non-classical secretion pathway could be the use of mass spectrometry followed by confirmation through specific antibodies.in silicoevidence, it should be experimentally confirmed before its exact clinical significance can be ascertained. Whether other troponin variants also enter the non-classical secretory pathway is also an open question. Nonetheless, it is fair to say that troponin non-classical secretion is a promising research field in cardiology that awaits to be explored.High-sensitivity assays have brought considerable uncertainty about cTn elevations. Troponin liberation due to non-AMI related causes remains elusive and its exact prognostic significance continues to be investigated. Since our proposal is based onFor additional information, please click here.


Introduction: Early and accurate Alzheimer’s disease (AD) diagnosis has evolved in recent years by the use of specific methods for detecting its histopathological features in concrete cases. Currently, biomarkers in cerebrospinal fluid (CSF) and imaging techniques (amyloid PET) are the most used specific methods. However, some results between both methods are discrepant. Therefore, an evaluation of these discrepant cases is required. Objective: The aim of this work is to analyze the characteristics of cases showing discrepancies between methods for detecting amyloid pathology. Methodology: Patients from the Neurology Department of La Fe Hospital (n = 82) were diagnosed using both methods (CSF biomarkers and amyloid-PET). Statistical analyses were performed using logistic regression, and sex and age were included as covariables. Additionally, results of standard neuropsychological evaluations were taken into account in our analyses. Results: The comparison between CSF biomarker (Aβ42) and amyloid PET results showed that around 18% of cases were discrepant—mainly CFS-negative and PET-positive cases had CSF levels close to the cut-off point. In addition, a correlation between the episodic memory test and CSF biomarkers levels was observed. However, the same results were not obtained for other neuropsychological domains. In general, CSF- and PET-discrepant cases showed altered episodic memory in around 66% of cases, while 33% showed normal performance. Conclusions: In common clinical practice at tertiary memory centers, result discrepancies between tests of amyloid status are far more common than expected. However, episodic memory tests remain an important support method for AD diagnosis, especially in cases with discrepant results between amyloid PET and CSF biomarkers. Increasingly, middle-aged people suffer from memory loss, subjective complaints without any medical evidence, cognitive impairment derived from other disorders , and dementia, with Alzheimer’s disease (AD) being the most common . AD diagRegarding the histopathological characteristics of AD, the extracellular formation of amyloid plaques and intracellular formation of neurofibrillary tangles are the main hallmarks; therefore, diagnostic criteria are based on these specific biomarkers. They are classified into amyloid status biomarkers (fibrillary amyloid retention by PET (amyloid PET), and Aβ42, t-Tau, and p-Tau in CSF) and neurodegeneration/topographic biomarkers  .The amyloid status biomarkers consist of CSF levels for Aβ42, t-Tau, and p-Tau, which provides relevant information for early AD diagnosis ,6. AnothThe recent development of CSF biomarkers and amyloid PET techniques in the early diagnosis of AD demonstrates important advances in the knowledge of the role of the amyloid status . HoweverOn the other hand, neuropsychological evaluation tests have shown their utility in the detection of neurodegenerative diseases, such as AD, and could constitute a useful tool to discern doubtful cases .In this sense, the present study is focused on the evaluation of the concordance between the results of CSF biomarkers levels  and amyloid PET in the diagnosis of patients with cognitive impairment to provide additional tools, such as neuropsychological tests, in order to help in discrepant case diagnosis in clinical practice.The study involved patients from the neurology unit from the Hospital Universitari I Politècnic la Fe  (n = 82), with ages between 48 and 80 years. All of them underwent two procedures for AD diagnosis: (i) lumbar puncture to obtain CSF samples for the determination of AD biomarkers (Amyloid β42 (Aβ42), t-Tau, and p-Tau); and (ii) amyloid PET technique . In addition, we evaluated all patients with neuropsychological tests  see .−1. The study protocol was approved by the Ethics Committee (CEIC) from the Health Research Institute La Fe . All participants signed the informed consent.We classified participants into two groups: (i) concordant cases (n = 67)—CSF biomarkers and amyloid PET imaging provided the same diagnosis; (ii) discordant cases (n = 15)—CSF biomarkers and amyloid PET imaging provided different diagnoses. We determined CSF positivity by Aβ42 levels with a cut off of 750 pg mLThe neuropsychological evaluation of the participants was based on the CDR and RBANS tests.The CDR test  establishes 5 possible states based on the evaluation of 6 areas of cognitive and behavioral autonomous functioning . The established states are: 0 , 0.5 (questionable dementia), 1 (mild dementia), 2 (moderate dementia), and 3 (severe dementia) .The RBANS  test is a neuropsychological battery that evaluates 5 areas: immediate memory (IM), language (L), attention (A), delayed memory (DM), and visuospatial construction (VC). A resulting score lower than 85 in some areas would indicate cognitive alteration ,17.We summarized participants’ demographic and clinical data using mean (standard deviation) and median (interquartile range) for numerical variables, and absolute frequency (%) for qualitative variables.We performed an evaluation of the relationship between the results of amyloid PET, biomarkers in CSF , and neuropsychological evaluation (RBANS and CDR) through logistic regression, including sex and age as covariates. We performed statistical analyses using R software version 4.0 (1) and clickR package version 0.4.39.−1). In addition, −1). Similarly, a comparison between amyloid PET and the ratio between t-Tau and Aβ42 showed 18% of discrepant cases.The diagnosis potential of the neuropsychological evaluation was evaluated with special attention to discordant cases; specifically, the scores obtained from CDR and RBANS. DM tests were assessed as complementary tools to the biochemical and imaging tests. For discrepant cases, patients with negative CSF biomarkers and positive PET manifest alterations in episodic memory in 60% of cases see . By cont−1, and most of them had high RBANS.DM scores ; however, discordant cases showed Aβ42 levels < 750 pg mL−1 and high RBANS.DM scores. Among positive PET cases, concordant cases showed Aβ42 levels < 750 pg mL−1, and most of them showed low RBANS.DM scores; furthermore, discordant cases showed Aβ42 levels > 750 pg mL−1 and low RBANS.DM scores.The relationship between Aβ42 levels and CSF and RBANS.DM tests is shown in At present, the gold standard for the diagnosis of AD is based on the evaluation of the amyloid status by means of CSF biomarker Aβ42 or amyloid PET imaging . In thisSometimes test results are inconclusive or lack agreement. Moreover, some studies showed discrepancies between both techniques ,12. AddiAccording to gender differences, we found higher discrepancies and higher positive results in females. The higher proportion of positive females could be explained by their higher risk levels for AD , as wellRegarding neuropsychology tests, they are mainly used to detect cognitive impairment cases and to evaluate disease progression . In thisIn the present study, the agreement between a CSF biomarker (Aβ42), and the amyloid PET technique was evaluated. The results showed that those subjects with alterations in amyloid PET (positive) could be AD patients; however, if CSF Aβ42 levels are ≥the cut-off point (negative), the AD diagnosis was not corroborated. In these discrepant cases, the patients’ cognitive impairment was evaluated according to the RBANS.DM domain, which constitutes an important tool in establishing diagnosis, much as it is in cases without studying amyloid status.An episodic memory assessment (RBANS.DM) would allow us to establish the phase of the disease , as the study by Hammers et al. already pointed out. In fact, they claimed that RBANS.DM could illuminate clinical decision making regarding the possible progression to dementia due to AD . In addiThe main limitations of this study are the small sample size and the reduced number of discordant cases. In addition, the participants were patients from the Neurology Unit; therefore, the sample could be biased. Moreover, the specific cut-off point used according to clinical practice in this unit could be different from others, making it difficult to extrapolate the results. Regarding amyloid PET, the interpretation of these results can be more subjective, especially in the most borderline cases. So, highly specialized personnel are required to be in charge of these tasks. Similarly, for neuropsychological evaluation, some specialized staff is required.Alzheimer’s disease diagnosis is mainly based on amyloid status (Aβ42 in CSF and amyloid PET). However, in some cases, both techniques presented discordant results. In these cases, classical complementary non-invasive and cost-effective neuropsychological tools continued providing the key data to support AD diagnosis. In this sense, episodic memory assessments still constitute a useful tool in supporting the diagnosis of patients with risks for Alzheimer’s disease development. Therefore, neuropsychological evaluations could help to increase knowledge regarding the patient’s cognitive impairment degree or disease progression but also identify AD patients.In general, this study highlights the high percentage of discrepancies between two techniques considered the gold standard in the diagnosis of AD. In this sense, there is an increasing need to carry out further research in plasma samples to identify new specific biomarkers that are minimally invasive and economically affordable.