Five years' experience of the clinical exome sequencing in a Spanish single center.

Nowadays, exome sequencing is a robust and cost-efficient genetic diagnostic tool already implemented in many clinical laboratories. Despite it has undoubtedly improved our diagnostic capacity and has allowed the discovery of many new Mendelian-disease genes, it only provides a molecular diagnosis in up to 25-30% of cases. Here, we comprehensively evaluate the results of a large sample set of 4974 clinical exomes performed in our laboratory over a period of 5 years, showing a global diagnostic rate of 24.62% (1391/4974). For the evaluation we establish different groups of diseases and demonstrate how the diagnostic rate is not only dependent on the analyzed group of diseases (43.12% in ophthalmological cases vs 16.61% in neurological cases) but on the specific disorder (47.49% in retinal dystrophies vs 24.02% in optic atrophy; 18.88% in neuropathies/paraparesias vs 11.43% in dementias). We also detail the most frequent mutated genes within each group of disorders and discuss, on our experience, further investigations and directions needed for the benefit of patients.

Automated assays for trace elements and ferritin measurement in saliva of pigs: Analytical validation and a pilot application to evaluate different iron status.

The aim of this study was to validate automated methods to measure iron (Fe), zinc (Zn), copper (Cu) and ferritin in pig saliva samples. A complete analytical validation was performed of all assays. In addition, these methods were applied to saliva of Fe supplemented (n = 22) and non-supplemented (n = 20) piglets. All assays were able to measure these biomarkers in pig saliva with adequate precision, accuracy and high sensitivity and, in case of trace elements without needing a deproteinization pre-process. The group of piglets supplemented with Fe presented significantly higher levels of ferritin and Zn in saliva. In conclusion, the automated assays evaluated were able to measure Fe, Zn, Cu and ferritin in saliva of pigs, and in case of trace elements, they have the advantage of not needing a deproteinization pre-treatment and thus these analytes can be measured in a simple and fast manner.

Biomarkers of sepsis in pigs, horses and cattle: from acute phase proteins to procalcitonin.

Sepsis is a complex clinical syndrome triggered by an inflammatory host response to an infection. It is usually complicated to detect and diagnose, and has severe consequences in human and veterinary health, especially when treatment is not started early. Therefore, efforts to detect sepsis accurately are needed. In addition, its proper diagnosis could reduce the misuse of antibiotics, which is essential fighting against antimicrobial resistance. This case is a particular issue in farm animals, as antibiotics have been traditionally given massively, but now they are becoming increasingly restricted. When sepsis is suspected in animals, the most frequently used biomarkers are acute phase proteins such as C-reactive protein, serum amyloid A and haptoglobin, but their concentrations can increase in other inflammatory conditions. In human patients, the most promising biomarkers to detect sepsis are currently procalcitonin and presepsin, and there is a wide range of other biomarkers under study. However, there is little information on the application of these biomarkers in veterinary species. This review aims to describe the general concepts of sepsis and the current knowledge about the biomarkers of sepsis in pigs, horses, and cattle and to discuss possible advances in the field.

Empiric treatment vs susceptibility-guided treatment for eradicating H. pylori: Is it possible to change that paradigm using modern molecular methods?

Helicobacter pylori (H. pylori) infection is the most widespread infectious-contagious disease worldwide, reaching a prevalence of 50-80% in developing countries. Chronic infection is considered the main cause of chronic gastritis and has been related to other diseases, such as peptic ulcer, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The most common treatment is with eradication regimens that utilize three or four drugs, including a proton pump inhibitor (PPI) and the antibiotics, clarithromycin and amoxycillin or metronidazole. Empiric antibiotic use for eradicating the bacterium has led to a growing resistance to those drugs, reducing regimen efficacy and increasing costs for both the patient and the healthcare sector. In such a context, the development of noninvasive next-generation molecular methods holds the promise of revolutionizing the treatment of H. pylori. The genotypic and phenotypic detection of the resistance of the bacterium to antibiotics enables personalized treatment regimens to be provided, reducing costs and implementing an antibiotic stewardship program. The aims of the present narrative review were to analyze and compare the traditional and next-generation methods for diagnosing H. pylori, explain the different factors associated with eradication failure, and emphasize the impact of the increasing antibiotic resistance on the reversal and prevention of H. pylori-associated diseases.

Basics for the potential use of saliva to evaluate stress, inflammation, immune system, and redox homeostasis in pigs.

The use of saliva as a biological sample has many advantages, being especially relevant in pigs where the blood collection is highly stressful and painful, both for the animal and the staff in charge of the sampling. Currently one of the main uses of saliva is for diagnosis and detection of infectious diseases, but the saliva can also be used to measure biomarkers that can provide information of stress, inflammation, immune response and redox homeostasis. This review will be focused on the analytes that can be used for such evaluations. Emphasis will be given in providing data of practical use about their physiological basis, how they can be measured, and their interpretation. In addition, some general rules regarding sampling and saliva storage are provided and the concept of sialochemistry will be addressed. There is still a need for more data and knowledge for most of these biomarkers to optimize their use, application, and interpretation. However, this review provides updated data to illustrate that besides the detection of pathogens in saliva, additional interesting applicative information regarding pigs´ welfare and health can be obtained from this fluid. Information that can potentially be applied to other animal species as well as to humans.

Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes.

Despite the improved accuracy of next-generation sequencing (NGS), it is widely accepted that variants need to be validated using Sanger sequencing before reporting. Validation of all NGS variants considerably increases the turnaround time and costs of clinical diagnosis. We comprehensively assessed this need in 1109 variants from 825 clinical exomes, the largest sample set to date assessed using Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing can be very useful as an internal quality control, but not so much as a verification method for high-quality single-nucleotide and small insertion/deletions variants. Laboratories might validate and establish their own thresholds before discontinuing Sanger confirmation studies. We also expand and validate 23 copy number variations detected by exome sequencing in 20 samples, observing a concordance of 95.65% (22/23).

Forty years of Tuberculous meningitis: The new face of an old enemy.

BACKGROUND: Tuberculous meningitis (TBM) occurs in 1-5% of cases of tuberculosis. Without early treatment, mortality and permanent disability rates are high. METHODS: A retrospective study performed at a tertiary hospital in Madrid (Spain) to describe clinical, diagnostic, and therapeutic aspects of TBM and analyze epidemiological trends over forty years, divided into two intervals (1979-1998 and 1999-2018). RESULTS: Overall, TBM was diagnosed in 65 patients (1.8% of new tuberculosis diagnoses), 48 in the first period and 17 in the second one. Median age at diagnosis increased from 38.5 to 77 years (p = 0.003). The proportion of non-HIV immunosuppressed patients increased (from 2.1% to 29.4%, p < 0.001), while the percentage of patients with a history of drug-abuse decreased (from 33.3% to 5.9%, p = 0.027). The median time between the onset of neurological symptoms and lumbar puncture increased from seven to 15 days (p = 0.040). The time between the onset of symptoms and the initiation of tuberculostatic treatment also increased from eleven to 18 days (p = 0.555). Results from image, biochemical, and microbiological tests showed no differences between both periods. A decreasing trend was observed in survival rates at 1-week (from 97.9% to 64.7%, p < 0.001), 1-month (from 91.7% to 58.8%, p = 0.002) and 1-year (from 85.4% to 47.1%, p = 0.002) after TBM diagnosis. CONCLUSIONS: The profile of patients diagnosed with TBM has changed from a young HIV-infected patient with a history of drug addiction to an elderly patient with non-HIV immunosuppression. Diagnosis and start of treatment both experienced a noticeable delay in the second period, which could help explain the increase in mortality observed across the two periods.

Salivary D-dimer in pigs: Validation of an automated assay and changes after acute stress.

D-dimer is a peptide found in serum and is derived from the degradation of blood clots. Even though it has been analysed in human saliva, D-dimer has not been previously evaluated in the saliva of any veterinary species, and its source and role remain unknown. The objectives of this research were firstly, to validate the use of an automated method for the measurement of D-dimer in porcine saliva, and secondly, to evaluate whether D-dimer concentration changes in pig saliva after an acute stress stimulus. For this purpose, a complete analytical validation of a commercially-available immunoturbidimetric assay was carried out. In addition, an experimental acute stress model was induced in 11 pigs based on a technique involving restraint by nose-snare immobilisation for 1 min. Saliva samples were subsequently collected at different times and D-dimer, salivary alpha-amylase (sAA) and cortisol were assessed in order to evaluate changes in its concentrations after the stress induction. The D-dimer automated assay showed adequate reproducibility and sensitivity, with coefficients of variation below 10% and a limit of quantification of 0.167 µg/mL fibrinogen equivalent units (FEU). It also showed a high accuracy, determined by linearity under dilution and recovery tests. In the stress model, a significant increase (P < 0.05) in salivary D-dimer 15 min after the stress stimulus and a positive correlation between D-dimer and sAA (r = 0.51; P < 0.001) were observed. These results indicate that D-dimer can be measured in porcine saliva with an automated method and suggest that its concentration can be influenced by stressful conditions.

[Primary central nervous system lymphoma: clinical experience in a neurological center]. / Linfoma primario del sistema nervioso central: experiencia clinica en un centro neurologico.

INTRODUCTION: Primary lymphoma of the central nervous system is a variety of non-Hodgkin's lymphoma that accounts for 4-5% of intracranial tumours and 5% of all lymphomas. It has its origin in the brain, the eyes, the leptomeninges and the spinal cord with no systemic evidence of lymphomatoid activity; the subtype of lymphoma is predominantly of B-type cells. PATIENTS AND METHODS: We conducted a descriptive study of the patients diagnosed with primary brain lymphoma who were attended to at third-level centres in Mexico between the years 1980 and 2016. Patients who had been screened for systemic lymphoma were included. The results were analysed by means of simple frequencies, and disease-free and overall survival time was analysed by Kaplan-Meier curves; the differences among curves were analysed by means of log rank. RESULTS: Of a total of 215 patients, there were only 74 cases. By sex, 45% were females and 55% were males. Regarding age, 36.7% were over 60 years old. The most frequent clinical manifestations were motor loss (60%) and cognitive disorders (52%). Most patients received some form of chemotherapy (89%). The only significant factor for radiological response and clinical prognosis was the combined use of radiochemotherapy (p = 0.04493). CONCLUSION: Lymphoma is a tumorous condition with a high clinicoradiological response to treatment, although the response is not long-lasting. Its early identification and multidisciplinary management are essential for a more favourable prognosis in these patients.

TITLE: Linfoma primario del sistema nervioso central: experiencia clinica en un centro neurologico.Introduccion. El linfoma primario del sistema nervioso central es una variedad de linfoma no Hodgkin que representa el 4-5% de los tumores intracraneales y el 5% de todos los linfomas. Se origina en el encefalo, los ojos, la leptomeninge y la medula espinal sin evidencia sistemica de actividad linfomatoide; el subtipo de linfoma mayoritariamente es de celulas de tipo B. Pacientes y metodos. Estudio descriptivo de los pacientes diagnosticados con linfoma cerebral primario que fueron atendidos en centros de tercer nivel en Mexico entre los años 1980 y 2016. Se incluyo a los pacientes que contaran con cribado para busqueda de linfoma sistemico. Los resultados se analizaron mediante frecuencias simples; en el caso del tiempo libre de enfermedad y supervivencia global, mediante curvas de Kaplan-Meier, y las diferencias entre curvas, mediante log rank. Resultados. En un total de 215 pacientes solo hubo 74 casos. El 45% fueron mujeres y el 55%, hombres. El 36,7% eran mayores de 60 años. Las manifestaciones clinicas mas frecuentes fueron deficit motor (60%) y alteraciones cognitivas (52%). La mayoria recibio alguna forma de quimioterapia (89%). El unico factor significativo para respuesta radiologica y pronostico clinico era el uso combinado de radioquimioterapia (p = 0,04493). Conclusion. El linfoma representa una patologia tumoral con alta respuesta clinicorradiologica al tratamiento, aunque la respuesta no es duradera. Es fundamental su identificacion temprana y el tratamiento multidisciplinario para el mejor pronostico de estos pacientes.

Radiotherapy plus temozolomide or PCV in patients with anaplastic oligodendroglioma 1p19q codeleted. / Radioterapia mas temozolomida o PCV en pacientes con oligodendroglioma anaplasico con codelecion 1p19q.

INTRODUCTION: Radiotherapy with procarbazine, lomustine, and vincristine (PCV) improves overall survival in patients with anaplastic oligodendroglioma 1p19q codeleted. PATIENTS AND METHODS: This retrospective analysis investigated outcomes in patients with anaplastic oligodendroglioma 1p19q codeleted compared two different protocols (radiotherapy plus temozolomide or PCV). The primary end points were overall survival and progression-free survival. Secondary endpoint was the radiological response. RESULTS: A total of 48 patients were included. Mean age was 43 years (range: 19-66 years), 26 were male (54.1%). Twenty-one patients received PCV and 27 temozolomide. The baseline characteristics were not difference between the groups. The progression-free survival and overall survival in the PCV group were 7.2 and 10.6 years respectively and temozolomide were 6.1 and 9.2 years, both statistically significant. The radiological response was present in 80.9% in PCV arm and 70.2% in temozolomide arm there was not statistical differences. The multivariate Cox model showed only the significant parameters the use of PCV protocol. The toxicity grade 3 or 4 was present in 42.8% in PCV arm and 11.1% in temozolomide arm. CONCLUSIONS: The most common strategy in the Latin America community is the substitution of the PCV for temozolomide. This retrospective study showed superior efficacy of PCV than temozolomide. The Latin American community effort must be made to be able to have the drugs to available for using as a first line of treatment.

TITLE: Radioterapia mas temozolomida o PCV en pacientes con oligodendroglioma anaplasico con codelecion 1p19q.Introduccion. La radioterapia con procarbacina, lomustina y vincristina (PCV) mejora la supervivencia global en pacientes con oligodendroglioma anaplasico con codelecion 1p19q, pero no esta disponible en America Latina. Pacientes y metodos. Analisis retrospectivo comparando dos protocolos diferentes, radioterapia mas temozolomida o PCV, en pacientes con oligodendroglioma anaplasico con codelecion 1p19q. Los objetivos primarios fueron la supervivencia global y la supervivencia libre de progresion, y el objetivo secundario, la respuesta radiologica. Resultados. Se incluyo a 48 pacientes, 26 de ellos varones (54,1%), con una edad media de 43 años (rango: 19-66 años). Veintiun pacientes recibieron PCV, y 27, temozolomida. Las caracteristicas iniciales no tuvieron diferencias entre los grupos. La supervivencia libre de progresion y la supervivencia global en el grupo con PCV fueron de 7,2 y 10,6 años, y en el grupo de temozolomida, de 6,1 y 9,2 años, respectivamente, unos resultados estadisticamente significativos. Hubo respuesta radiologica en el 80,9% en el brazo de PCV y el 70,2% en el brazo de temozolomida. El analisis multivariado de Cox mostro como unico parametro significativo el uso del protocolo PCV. El grado de toxicidad 3-4 estuvo presente en el 42,8% en el brazo de PCV y en el 11,1% en el brazo de temozolomida. Conclusiones. La estrategia mas comun en America Latina es la sustitucion de PCV por temozolomida. Este estudio retrospectivo mostro una eficacia superior de PCV que de la temozolomida. La diferencia obliga a la comunidad latinoamericana a hacer un esfuerzo colectivo para poder tener acceso a los medicamentos para su uso como primera linea de tratamiento.

Formación en anestesia regional guiada por ultrasonidos / Training in regional anaesthesia

Programa de formación de médicos residentes en anestesia regional guiada por ecografía. Cronograma y objetivos (AU)

Guidelines for the training of residents in the domain of regional anaesthesia with ecography. Objetives and development (AU)

Influence of ionic strength on the flexibility of alginate studied by size exclusion chromatography.

SEC measurements of the [η]-M relationship for alginate from Macrocystis pyrifera and the wormlike model can be used to characterize flexibility through two independent treatments (Bohdanecky's equations and HYDFIT program), both providing the same results. Two different assumptions concerning mass per unit of length lead to different conclusions. First: persistence length decreases with ionic strength (the intrinsic component of the persistence length is 11.3 nm and the electrostatic component is 6 nm when ionic strength is 0.01). Second: persistence length is independent of ionic strength (12 nm). Either of these options shows that the wormlike model in itself is not sufficient to explain flexibility over the whole range of chain lengths for these polyelectrolytes. A plausible explanation could be the presence of a combination of short-range and long-range screening effects of the ions of the solutions. This would also explain some data found in the literature regarding alginate flexibility.

Semblanza del Dr. José Rafael Estrada González (1921–1991) / Biographical sketch of Dr. José Rafael Estrada González (1921–1991)

Se realiza una semblanza del Dr. José Rafael Estrada González (23.08.1921 a 31.03.1991), maestro y creador de la“Escuela Neurológica Cubana”(AU)

A semblance was made about Dr. José Rafael Estrada González (23.08.1921 to 31.03.1991), teacher and creator of “Cuban Neurological School”(AU)

El Dr. Manuel González Echeverría en la historia de la epilepsia / Dr. Manuel González Echeverría in epilepsy history

Se realiza una cronología de la obra del insigne neuropsiquiatra Dr. Manuel González Echeverría (23.04.1833 a26.03.1898), y especialmente su abnegada y laboriosa dedicación al estudio de la epilepsia(AU)

A chronology was made about the work of distinguished neuropsychiatric Dr. Manuel González Echeverría (23.04.1833 to 26.03.1898), and especially his abnegate and laborious dedication in epilepsy study(AU)

Frequency of ABCA4 mutations in 278 Spanish controls: an insight into the prevalence of autosomal recessive Stargardt disease.

AIM: To determine the carrier frequency of ABCA4 mutations in order to achieve an insight into the prevalence of autosomal recessive Stargardt disease (arSTGD) in the Spanish population. METHODS: arSTGD patients (n = 133) were analysed using ABCR400 microarray and sequencing. Control subjects were analysed by two different strategies: 200 individuals were screened for the p.Arg1129Leu mutation by denaturing-HPLC and sequencing; 78 individuals were tested for variants with the microarray and sequencing. RESULTS: For the first strategy in control subjects, the p.Arg1129Leu variant was found in two heterozygous individuals, which would mean a carrier frequency for any variant of approximately 6.0% and a calculated arSTGD prevalence of 1:1000. For the second strategy, carrier frequency was 6.4% and therefore an estimated prevalence of the disease of 1:870. CONCLUSION: Calculated prevalence of arSTGD based on the ABCA4 carrier frequency could be considerably higher than previous estimation. This discrepancy between observed (genotypic) and estimated (phenotypic) prevalence could be due to the existence of non-pathological or low penetrance alleles, which may result in late-onset arSTGD or may be implicated in age-related macular degeneration. This situation should be regarded with special care when genetic counselling is given and further follow-up of these patients should be recommended.