Detection of cerebrospinal fluid biomarkers changes of Alzheimer's disease using a cognitive stress test in persons with subjective cognitive decline and mild cognitive impairment.

Introduction: Timely and accurate diagnosis of the earliest manifestations of Alzheimer's disease (AD) is critically important. Cognitive challenge tests such as the Loewenstein Acevedo Scales for Semantic Interference and Learning (LASSI-L) have shown favorable diagnostic properties in a number of previous investigations using amyloid or FDG PET. However, no studies have examined LASSI-L performance against cerebrospinal fluid biomarkers of AD, which can be affected before the distribution of fibrillar amyloid and other changes that can be observed in brain neuroimaging. Therefore, we aimed to evaluate the relationship between LASSI-L scores and CSF biomarkers and the capacity of the cognitive challenge test to detect the presence of amyloid and tau deposition in patients with subjective cognitive decline and amnestic mild cognitive impairment (MCI). Methods: One hundred and seventy-nine patients consulting for memory loss without functional impairment were enrolled. Patients were examined using comprehensive neuropsychological assessment, the LASSI-L, and cerebrospinal fluid (CSF) biomarkers (Aß1-42/Aß1-40 and ptau181). Means comparisons, correlations, effect sizes, and ROC curves were calculated. Results: LASSI-L scores were significantly associated with CSF biomarkers Aß1-42/Aß1-40 in patients diagnosed with MCI and subjective cognitive decline, especially those scores evaluating the capacity to recover from proactive semantic interference effects and delayed recall. A logistic regression model for the entire sample including LASSI-L and age showed an accuracy of 0.749 and an area under the curve of 0.785 to detect abnormal amyloid deposition. Conclusion: Our study supports the biological validity of the LASSI-L and its semantic interference paradigm in the context of the early stages of AD. These findings emphasize the utility and the convenience of including sensitive cognitive challenge tests in the assessment of patients with suspicion of early stages of AD.

A data-driven approach to complement the A/T/(N) classification system using CSF biomarkers.

AIMS: The AT(N) classification system not only improved the biological characterization of Alzheimer's disease (AD) but also raised challenges for its clinical application. Unbiased, data-driven techniques such as clustering may help optimize it, rendering informative categories on biomarkers' values. METHODS: We compared the diagnostic and prognostic abilities of CSF biomarkers clustering results against their AT(N) classification. We studied clinical (patients from our center) and research (Alzheimer's Disease Neuroimaging Initiative) cohorts. The studied CSF biomarkers included Aß(1-42), Aß(1-42)/Aß(1-40) ratio, tTau, and pTau. RESULTS: The optimal solution yielded three clusters in both cohorts, significantly different in diagnosis, AT(N) classification, values distribution, and survival. We defined these three CSF groups as (i) non-defined or unrelated to AD, (ii) early stages and/or more delayed risk of conversion to dementia, and (iii) more severe cognitive impairment subjects with faster progression to dementia. CONCLUSION: We propose this data-driven three-group classification as a meaningful and straightforward approach to evaluating the risk of conversion to dementia, complementary to the AT(N) system classification.

Integration of Multiple Interferometers in Highly Multiplexed Diagnostic KITs to Evaluate Several Biomarkers of COVID-19 in Serum.

In the present work, highly multiplexed diagnostic KITs based on an Interferometric Optical Detection Method (IODM) were developed to evaluate six Coronavirus Disease 2019 (COVID-19)-related biomarkers. These biomarkers of COVID-19 were evaluated in 74 serum samples from severe, moderate, and mild patients with positive polymerase chain reaction (PCR), collected at the end of March 2020 in the Hospital Clínico San Carlos, in Madrid (Spain). The developed multiplexed diagnostic KITs were biofunctionalized to simultaneously measure different types of specific biomarkers involved in COVID-19. Thus, the serum samples were investigated by measuring the total specific Immunoglobulins (sIgT), specific Immunoglobulins G (sIgG), specific Immunoglobulins M (sIgM), specific Immunoglobulins A (sIgA), all of them against SARS-CoV-2, together with two biomarkers involved in inflammatory disorders, Ferritin (FER) and C Reactive Protein (CRP). To assess the results, a Multiple Linear Regression Model (MLRM) was carried out to study the influence of IgGs, IgMs, IgAs, FER, and CRP against the total sIgTs in these serum samples with a goodness of fit of 73.01% (Adjusted R-Squared).

Relación entre la cuantificación de calcio coronario y la composición de la microbiota intestinal en sujetos sin enfermedad cardiovascular previa: estudio piloto / Relationship between the coronary artery calcium quantification and gut microbiota composition in subjects without previous cardiovascular disease: A pilot study

Objetivo: Determinar si existe alguna asociación entre el perfil de microbiota intestinal y la carga aterosclerótica global medida mediante cuantificación de calcio coronario (CCC) en sujetos sin antecedentes de enfermedad cardiovascular (ECV). Métodos: Se incluyeron 20 pacientes mayores de edad, sin antecedentes de ECV a los que se les hizo la CCC mediante tomografía computarizada multicorte. Además, se les recogió una muestra de heces para caracterizar la composición de la microbiota intestinal mediante la secuenciación del gen 16S RNAr con técnicas de secuenciación masiva y una muestra de sangre para la cuantificación de citoquinas proinflamatorias, mediante ELISA específicos, y del metabolito bacteriano N-óxido de trimetilamina (TMAO) por cromatografía de gases/líquidos acoplada a espectrometría de masas en tándem. Resultados: La media de edad fue de 63,5 años y un 60% eran mujeres. La mitad de los pacientes (n=10) presentaron una CCC>100 y se caracterizaron por una mayor abundancia de bacterias del filo Proteobacteria, pertenecientes principalmente a las familias Enterobacteriaceae y Pasteurellaceae, que los pacientes con CCC≤100. La mayoría de los géneros bacterianos identificados, Enterobacter, Escherichia/Shigella, Klebsiella, Citrobacter y Salmonella, se asociaron positivamente con los niveles plasmáticos de TNF-α o IL-1β y con la producción de TMAO. Conclusión: Los resultados de este estudio piloto muestran un perfil de microbiota intestinal asociado a la presencia de CCC>100 en pacientes sin ECV previa, caracterizado por un aumento en la proporción de géneros bacterianos productores de TMAO. Puesto que la composición de la microbiota intestinal es altamente modulable por diversos factores, es posible que, en un futuro, podamos prevenir, e incluso intervenir, la enfermedad cardiovascular mediante estrategias nutricionales. (AU)

Aim: To investigate the relationship between gut microbiota composition and the presence of coronary atherosclerosis assessed by coronary artery calcium (CAC) quantification in individuals without previous cardiovascular disease (CVD). Methods: We included 20 patients over 18 years of age with no history of CVD who underwent multiple detector-computed tomography. From each patient, a stool sample was obtained to characterize gut microbiota composition by sequencing bacterial 16S ribosomal RNA gene. In addition, circulating levels of TNF-α and IL-1β, as well as trimethylamine N-oxide (TMAO) were determined in plasma samples by automated ELISA and capillary gas chromatography-mass spectrometry, respectively. Results: The mean age of patients was 63.5 years and 60% were women. Half of patients had CAC >100 (Agatston score), and were characterized by a higher abundance of the phylum Proteobacteria, mainly of bacteria belonging to the families Enterobacteriaceae and than patients with a CAC ≤ 100. Moreover, bacterial genera identified as biomarkers, such as Enterobacter, Escherichia/Shigella y Klebsiella, were positively associated with inflammation levels and with TMAO production. Conclusion: Our data shows a gut microbiota profile associated with the presence of coronary calcium in patients without previous CVD. Although there are no strategies to decrease the amount of coronary calcium, gut microbiota is highly malleable by several factors. The possibility of preventing and even intervening CVD progression through strategies targeted gut microbiota is a very attractive idea that deserves further studies. (AU)

Relationship between the coronary artery calcium quantification and gut microbiota composition in subjects without previous cardiovascular disease: A pilot study. / Relación entre la cuantificación de calcio coronario y la composición de la microbiota intestinal en sujetos sin enfermedad cardiovascular previa: estudio piloto.

AIM: To investigate the relationship between gut microbiota composition and the presence of coronary atherosclerosis assessed by coronary artery calcium (CAC) quantification in individuals without previous cardiovascular disease (CVD). METHODS: We included 20 patients over 18 years of age with no history of CVD who underwent multiple detector-computed tomography. From each patient, a stool sample was obtained to characterize gut microbiota composition by sequencing bacterial 16S ribosomal RNA gene. In addition, circulating levels of TNF-α and IL-1ß, as well as trimethylamine N-oxide (TMAO) were determined in plasma samples by automated ELISA and capillary gas chromatography-mass spectrometry, respectively. RESULTS: The mean age of patients was 63.5 years and 60% were women. Half of patients had CAC >100 (Agatston score), and were characterized by a higher abundance of the phylum Proteobacteria, mainly of bacteria belonging to the families Enterobacteriaceae and than patients with a CAC ≤ 100. Moreover, bacterial genera identified as biomarkers, such as Enterobacter, Escherichia/Shigella y Klebsiella, were positively associated with inflammation levels and with TMAO production. CONCLUSION: Our data shows a gut microbiota profile associated with the presence of coronary calcium in patients without previous CVD. Although there are no strategies to decrease the amount of coronary calcium, gut microbiota is highly malleable by several factors. The possibility of preventing and even intervening CVD progression through strategies targeted gut microbiota is a very attractive idea that deserves further studies.

"Brain Fog" by COVID-19 or Alzheimer's Disease? A Case Report.

Cognitive symptoms after COVID-19 have been increasingly recognized several months after the acute infection and have been designated as "brain fog." We report a patient with cognitive symptoms that started immediately after COVID-19, in which cerebrospinal fluid biomarkers were highly suggestive of Alzheimer's disease. Our case highlights the need to examine patients with cognitive symptoms following COVID-19 comprehensively. A detailed assessment combining clinical, cognitive, and biomarker studies may help disentangle the underlying mechanisms associated with cognitive dysfunction in each case. The investigation of neurodegenerative processes in an early stage, especially in older patients, is probably warranted.

Herpesvirus Antibodies, Vitamin D and Short-Chain Fatty Acids: Their Correlation with Cell Subsets in Multiple Sclerosis Patients and Healthy Controls.

Although the etiology of multiple sclerosis (MS) is still unknown, it is commonly accepted that environmental factors could contribute to the disease. The objective of this study was to analyze the humoral response to Epstein-Barr virus, human herpesvirus 6A/B and cytomegalovirus, and the levels of 25-hydroxyvitamin D (25(OH)D) and the three main short-chain fatty acids (SCFA), propionate (PA), butyrate (BA) and acetate (AA), in MS patients and healthy controls (HC) to understand how they could contribute to the pathogenesis of the disease. With this purpose, we analyzed the correlations among them and with different clinical variables and a wide panel of cell subsets. We found statistically significant differences for most of the environmental factors analyzed when we compared MS patients and HC, supporting their possible involvement in the disease. The strongest correlations with the clinical variables and the cell subsets analyzed were found for 25(OH)D and SCFAs levels. A correlation was also found between 25(OH)D and PA/AA ratio, and the interaction between these factors negatively correlated with interleukin 17 (IL-17)-producing CD4+ and CD8+ T cells in untreated MS patients. Therapies that simultaneously increase vitamin D levels and modify the proportion of SCFA could be evaluated in the future.

Predictive factors and early biomarkers of response in multiple sclerosis patients treated with natalizumab.

There are an increasing number of treatments available for multiple sclerosis (MS). The early identification of optimal responders to individual treatments is important to achieve individualized therapy. With this aim, we performed a multicenter retrospective longitudinal study including 186 MS patients treated with natalizumab who were followed for 2 years. We analyzed the following variables at recruitment: sex, current age, age at disease onset, disease duration, EDSS, number of T2 and Gd + lesions, IgG and IgM oligoclonal bands, HLA class II (DR, DRB, DQA, DQB, and DRB1*15:01), IgG and IgM antibody titers against human herpesvirus 6 (HHV-6) and the antibody response to Epstein-Barr virus (EBV) through the measurement of the anti-EBNA-1 and anti-VCA IgG titers, in relation to clinical response (no relapses or disability progression), and to NEDA-3 (no evidence of disease activity in terms of clinical response and no changes in MRI scans either) after 2-years follow-up. Baseline EDSS score, baseline EBNA-1 IgG titers and percentage change of HHV6 IgG titers between baseline and 6 month visits were significantly different in clinical responders and in NEDA-3 status (all of them remained significant in the multivariate analysis). We identified three variables for the early identification of natalizumab optimal responders in a rapid and cost-effective approach.

Syncytin-1/HERV-W envelope is an early activation marker of leukocytes and is upregulated in multiple sclerosis patients.

Syncytin-1 is the envelope protein of the human endogenous retrovirus W (HERV-W). It has been related to multiple sclerosis (MS) but its role in cellular immunity and its pathogenic mechanism in the autoimmune context are not fully understood. We analyzed syncytin-1 levels in peripheral blood mononuclear cells (PBMC) subsets from healthy donors, MS patients in relapse or remission, and patients with acute infections by flow cytometry. PBMC cultures were also prepared to analyze protein expression kinetics. MS patients had higher levels of syncytin-1 levels than controls. We found that syncytin-1 is elevated in monocytes during MS relapses and infections. Cells expressing syncytin-1, including monocytes, T and B lymphocytes, and NKs presented mainly an activated phenotype and, upon stimulation with LPS, its levels increased rapidly on antigen-presenting cells. Syncytin-1 ligation promoted the activation of monocytes, as demonstrated by the upregulation of CD80 and the nonclassical subset CD14low CD16+ . Our results suggest an important role for syncytin-1 in the activation of leukocytes. Given that the expression of syncytin-1 is upregulated in MS patients, this protein might be contributing to the autoimmune cascade in the disease.

Development and validation of an HPLC-MS method for a simultaneous measurement of oxalate and citrate in urine / Desarrollo y validación de un método de HPLC-MS para la medida simultánea de oxalato y citrato en orina

Objectives. An increased urinary oxalate and reduced urinary citrate are considered major risk factors in the formation of calcium oxalate kidney stones. In this work, an HPLC-MS method is presented for the simultaneous measurement of oxalate and citrate in urine. Methods. Sample preparation was carried out using a liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed in a C18 column by gradient elution with methanol and 1 M formate buffer at 35 °C. Citrate and oxalate were monitored on a single-quadrupole MS system. Results. The method was linear in the concentration range of 0.5 mg/L to 450 mg/L for oxalate and from 2.5 mg/L to 950 mg/L for citrate. The Lower Limit of Measurement was 0.56 mg/L for oxalate and 2.5 mg/L for citrate. The within-day imprecision was 6% for oxalate and 3% for citrate, and the between day imprecision was lower than 15% for both analytes. LC-MS method was compared with capillary electrophoresis and it was shown that both methods were interchangeable to measure oxalate, but not citrate. Conclusions. HPLC-MS method is a good approach to measure oxalate and citrate in 24-hour urine, and it is applicable in clinical routine for patients with recurrent stone formation (AU)

Objetivos. La hiperoxaluria e hipocitraturia están consideradas el principal factor de riesgo en la formación de cálculos renales de oxalato cálcico. En este trabajo se ha desarrollado un método de HPLC-MS para la medida simultánea de oxalato y citrato en orina. Métodos. La preparación de muestras se realizó por una extracción líquido-líquido con etilacetato. La separación cromatográfica se llevó a cabo en una columna C-18 a 35 °C con un gradiente de elución con metanol y ácido fórmico 1 M. El citrato y el oxalato se monitorizaron mediante espectrometría de masas con un cuadrupolo simple. Resultados. El método fue lineal para el oxalato en el rango de concentración de 0,5 a 450 mg/l y para el citrato de 2,5 a 950 mg/l. El límite menor de intervalo de medida fue de 0,56 mg/l para el oxalato y de 2,5 mg/l para el citrato. La imprecisión intradía fue del 6% para el oxalato y del 3% para el citrato, y la interdía fue inferior al 15% para ambos analitos. El método de LC-MS desarrollado se comparó con un método de electroforesis capilar y se demostró que ambos eran intercambiables para el oxalato, pero no para el citrato. Conclusiones. El método de HPLC-MS desarrollado constituye una buena aproximación para medir oxalato y citrato en orina de 24 horas y es aplicable en la rutina clínica para pacientes con riesgo de formación de cálculos renales (AU)

Anti-human herpesvirus 6A/B IgG correlates with relapses and progression in multiple sclerosis.

OBJECTIVE: To analyze the titers of the IgG and IgM antibodies against human herpesvirus 6A/B (HHV-6A/B) in multiple sclerosis (MS) patients treated with different disease modified therapies (DMTs) along two-years of follow-up. METHODS: We collected 2163 serum samples from 596 MS; for 301 MS patients a 2-years follow-up was performed. Serum samples of 337 healthy controls were also analyzed. Anti-HHV-6A/B IgG and IgM were analyzed by ELISA (Panbio). RESULTS: We found that 129/187 (69.0%) MS patients with a decrease of the anti-HHV-6A/B IgG titers after 2-years with DMTs were free of relapses and progression vs. 46/113 (40.7%) of MS patients with an increase of the anti-HHV-6A/B IgG titers (p = 0.0000015); the higher significance was found for natalizumab. Furthermore, we found that anti-HHV-6A/B IgG titers reached their highest value two weeks before the relapse (p = 0.0142), while the anti-HHV-6A/B IgM titers reached their highest value one month before the relapse (p = 0.0344). CONCLUSION: The measurement of the anti-HHV-6A/B IgG titers could be a good biomarker of clinical response to the different DMTs. The increase of the anti-HHV-6A/B IgG and IgM titers predicts the upcoming clinical relapses. However, further longitudinal studies are needed to validate these results.

La seguridad del paciente en el laboratorio clínico. Implantación de un protocolo de identificación inequívoca de paciente / Patient safety in the clinical laboratory. introduction of a patient identification procedure

Los errores en la identificación de paciente representan uno de los problemas con mayor índice de factor de riesgo en provocar un evento adverso en el paciente. Objetivo. Implantar una estrategia de mejora para la identificación de paciente y sus muestras biológicas en las solicitudes de pruebas diagnósticas. Material y métodos. Diseño del proceso y protocolo a seguir por todos los profesionales implicados en el proceso de petición y realización de pruebas analíticas. Elaboración de una normativa de seguridad de identificación de paciente y sus muestras biológicas, de obligado cumplimiento. Evaluación de indicadores antes y después de la implantación de dichas medidas de mejora. Resultados. Se observó una disminución del % de incidencias en la cumplimentación de los datos de paciente antes y después de la implantación de las medidas de mejora (5,70% versus 0,97% en urgentes y 21,5% versus 2,09% en hospitalización). Sin embargo, no se observó una disminución significativa en los casos en los que la identificación del paciente se realizó de forma manual (6,9% versus 5,3% en urgentes y 8,3% versus 9,5% en hospitalización). Conclusiones. Las estrategias de mejora aplicadas han demostrado ser eficaces en la mejora de la identificación del paciente en las solicitudes analíticas. No obstante, debemos seguir trabajando en esta estrategia, fomentando la cultura de seguridad de todos los profesionales implicados e intentando alcanzar el objetivo de que el 100% de las solicitudes estén identificadas correctamente (AU)

Errors in patient identification represent one of the problems with risk factor index in causing an adverse event in the patient. Objective. Development of Improvement Strategies for the identification of patients and their biological samples. Material and methods. Design of the Procedure and Protocol to follow by all the professionals involved in the request process and production of analytical tests. Preparation of Safety Guidelines for the identification of patients and their biological samples. Evaluation of Indicators before and after the introduction of these improvement strategies. Results. Decreases in the number of incidents were observed after introducing the improved procedures; 5.70% versus 0.97% in emergencies, and 21.5% versus 2.09% in the hospital patients. However, no significant decreases were observed in cases where patient identification was made manually; 6.9% versus 5.35% in emergencies and 8.3% versus 9.5% in hospital patients. Conclusions. The improvement strategies applied have demonstrated to be effective in improving the identification of the patient in the analytical requests. However, we must continue working with this strategy, encouraging the safety culture in all the professionals involved, and try to achieve objective of 100% of the requests being identified correctly (AU)

Falsos negativos en la detección de componentes monoclonales por electroforesis capilar / False negatives in the analysis of monoclonal components by capillary electrophoresis

La electroforesis capilar es una técnica rápida y automatizada que permite la detección de componentes monoclonales en suero con alta sensibilidad, estando ampliamente instaurada en los laboratorios clínicos. Se han descrito sin embargo, falsos positivos y negativos que plantean problemas a la hora de interpretar el trazado electroforético. Los falsos positivos son debidos fundamentalmente a sustancias exógenas no proteicas presentes en la muestra que absorben radiación en la región ultravioleta dando lugar a picos anómalos en el proteinograma. Los falsos negativos se deben en su mayoría a la baja concentración del componente monoclonal, aunque se han descrito también cuando se encuentran en alta concentración, debidos principalmente a las propiedades físico-químicas de la paraproteína que provocan una separación incorrecta de la misma. Presentamos una serie de casos con componentes monoclonales de tipo IgA e IgM, detectados inicialmente por electroforesis de acetato de celulosa y presentes en la muestra en alta concentración, en los que la electroforesis capilar tuvo problemas para su detección (AU)

Capillary electrophoresis is a fast, automated and highly sensitive technique for the detection of monoclonal components in serum that is being increasingly introduced in clinical laboratories. Nevertheless, false negative and positive results have been reported, and thus the electrophoretic pattern may be difficult to interpret. False positive results are chiefly due to exogenous substances other than proteins present in serum, which absorb at UV wavelengths and can produce an abnormal spike. False negative results are mainly due to very low concentrations of monoclonal components. However, high concentration monoclonal components may not be correctly detected due to the physicochemical properties of the paraproteins, which may cause anomalous separation. In this work, we study three cases with high concentration monoclonal components of the IgA and IgM classes. They were initially detected on cellulose acetate electrophoresis, but the capillary electrophoresis was not able to correctly detect them (AU)