Hallazgos histopatológicos en pacientes con síndrome de ojo seco secundario a enfermedad autoinmune tratados con suero autógeno

ResumenObjetivoDeterminar las características histopatológicas en biopsia de conjuntiva bulbar en pacientes con síndrome de ojo seco tratados con suero autógeno y carboximetilcelulosa.Materiales y métodosSe reclutaron pacientes con diagnóstico de síndrome de ojo seco. Se les sometió a toma de biopsia de conjuntiva bulbar de ambos ojos. Se indicó a cada paciente tratamiento con suero autógeno al 20% una gota 4 veces al día en ojo derecho y carboximetilcelulosa una gota 4 veces al día en ojo izquierdo durante un mes, tras lo cual se tomó una segunda biopsia de conjuntiva bulbar. Se realizó estudio histopatológico de cada biopsia, considerando grado de infiltrado inflamatorio, grado de metaplasia escamosa y número de células caliciformes, dividiendo mediante escala numérica las variables mencionadas en 4 grados y comparando resultados con prueba de Wilcoxon.ResultadosSe incluyeron dentro del protocolo 32 ojos pertenecientes a 16 pacientes, con una edad media de 51.89 años. Los hallazgos histopatológicos posteriores al tratamiento con suero autógeno fueron: en 12 de los pacientes no hubo cambios en el grado de metaplasia escamosa, y en 4 disminuyó (p=0.046); en 5 pacientes se incrementó el número de células caliciformes (p=0.049), en 10 no se modificó y en uno disminuyó; el grado de infiltrado inflamatorio aumentó en 6 (p=0.014) pacientes y no se modificó en 10. Con carboximetilcelulosa, la metaplasia disminuyó en 2 pacientes, aumentó en uno y se mantuvo igual en el resto; el número de células caliciformes aumentó en 6 pacientes, disminuyó en 6 y no se modificó en 4; el infiltrado inflamatorio aumentó en 4 pacientes, disminuyó en 2 y se mantuvo igual en el resto.ConclusionesEl tratamiento con suero autógeno ofrece una mejoría estadísticamente significativa en cuanto al aumento de células caliciformes conjuntivales y a la disminución del grado de metaplasia escamosa. En comparación, con el tratamiento con carboximetilcelulosa no se observaron cambios estadísticamente significativos después del tratamiento en ninguna de las variables.AbstractPurposeTo determine histopathological features in conjunctival biopsy of patients with dry eye syndrome treated with autologous serum and carboximethilcelulose.Materials and methodsConjunctival biopsy was taken from both eyes of patients with dry eye syndrome. Right eyes were treated with 20% autologous serum one drop qid, whereas left eyes received carboximethilcelulose one drop qid for one month, after which new biopsies were taken. Histopathological analysis was performed, taking into account the degree of inflammatory infiltrate, degree of squamous metaplasia, and the number of goblet cells, dividing those variables in a 4 degree scale and comparing results with Wilcoxon test.ResultsA total of 32 eyes of 16 patients were included (mean age 51.89 years). After treatment with autologous serum, 12 patients showed no change in the degree of squamous metaplasia and it diminished in 4 (P=.046); 5 showed an increased number of goblet cells (P=.049), in one patient the number diminished and 10 showed no change; the inflammatory infiltrate increased in 6 patients (P=.014) and showed no change in the rest. After treatment with carboximethilcelulose, squamous metaplasia diminished in 2 patients, increased in one and the rest showed no change; the number of goblet cells increased in 6, was reduced in 6 and 4 showed no change; the inflammatory infiltrate increased in 4, was reduced in 2 and the rest showed no change.ConclussionTreatment with autologous serum offers a statistically significant improvement regarding the number of goblet cells and squamous metaplasia. Treatment with carboximethilcelulose showed no significant change in any of the variables

Molecular Diagnosis of Invasive Aspergillosis

Invasive aspergillosis (IA) is a disease that is difficult to manage and is associated with a significantly high morbidity and mortality, caused by different species of the genus Aspergillus, and closely related to immunocompromised patients; thus, it is important to understand the distribution and molecular epidemiology of the species causing this disease. Even though Aspergillus fumigatus sensu stricto is the most common species that cause IA, in recent years, there has been an increase in the number of species in the different sections which makes the diagnosis of this invasive fungal disease a great challenge. Conventional tests for the diagnosis of IA present limitations in sensitivity and specificity, while molecular tests have the potential to improve diagnosis by offering a more sensitive and rapid identification, but they are not yet standardized for reliable use in clinic. Nevertheless, there are some tests for the presumptive diagnosis of aspergillosis which, although are not specific for the identification of species, have been decisive in the case of IA. Among these are the Galactomannan test (GM), the Beta-D-glucan assay and volatile organic compounds (VOCs) testing. In this chapter, the recent advances and challenges in the molecular diagnosis of IA are revised

Genotyping of Leptospira interrogans isolates from Mexican patients

The aim of this study was genotypically characterize Leptospira sp. clinical isolates from Mexico which were previously identified as Leptospira interrogans serovar Pomona (POM) by phenotypic methods. The Random Amplified Polymorphic DNA (RAPD) method was used for DNA amplification with five oligonucleotides. A dendrogram was constructed using the Unweighted Pair Group Method Analysis (UPGMA). During the genotypic characterization, the studied isolates constituted a group which was associated with the reference strain L. interrogans serovar Pomona. The Minimum Spanning Networks (MST) analysis revealed the same cluster between Mexican isolates and the reference strain POM. Clinical isolates identified as L. interrogans serovar POM have a clonal reproduction type, suggesting that this clone is distributed in different regions of Mexico

Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates

Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto, C. nivariensis and C. bracarensis. In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto, C. nivariensis, and C. bracarensis, respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolates. Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences, and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species

Genotyping of clinical isolates of Aspergillus flavus and its relationship with environmental isolates center

Antecedentes: Durante un período de 4 meses, y mientras se llevaba a cabo un muestreo ambiental de aire, se diagnosticaron 2 casos de aspergilosis por Aspergillus flavus en un centro oncohematológico de Buenos Aires, Argentina. Objetivos: Conocer la variabilidad y la relación genética entre los aislamientos clínicos y los ambientales obtenidos en el centro oncohematológico. Métodos: Se utilizaron 2 técnicas de genotipificación con diferente poder discriminatorio (RAPD y AFLP). Una matriz de similitud genética fue calculada usando el método de Jaccard y fue la base para la cons- trucción de un dendrograma por el método de UPGMA. Se estimó el nivel de variabilidad genética por medio del porcentaje de loci polimórficos, número de alelos efectivos y heterocigosidad esperada, y el índice de asociación (I A ) . Resultados: El dendrograma mostró que los aislamientos de A. flavus recuperados de los pacientes no se relacionaron genéticamente con los del ambiente nosocomial. Los valores más altos de diversidad gené- tica correspondieron a los aislamientos ambientales. El I A estimado para todos los aislamientos sugiere eventos de recombinación. Conclusiones: Los pacientes 1 y 2 no fueron infectados con los aislamientos obtenidos del ambiente hospitalario. Los aislamientos clínicos y ambientales de A. flavus mostraron alta variabilidad genética entre ellos

Vertidos tóxicos al río Guadiamar: propuestas técnicas para su corrección

Inmediatamente de producirse el vertido tóxico al río Guadiamar, el Grupo T.A.R. se lanzó sin pensarlo dos veces a la búsqueda de soluciones técnicas a un panorama desolador y de efectos desconocidos, todos ellos amenazantes. El ácido “se comía el suelo inundado” por la riada, el agua retenida en Entremuros a pH 3, y con un enorme contenido de metales pesados, ocupaba una extensión de kilómetros. Nos hundimos en el agua hasta el cuello, y cuando nos cubría cogimos la barca, metimos el río a pedazos en nuestro laboratorio, para trabajar todas las hipótesis, ensayar todas las posibilidades. Peleando con la realidad le sacamos datos al Guadiamar, diseñamos actuaciones, poniéndole ingeniería a cuantas hipótesis nos planteaba la situación. En primera fila observamos las mejores actuaciones que nadie diseñó. El propio río, activando sus defensas naturales, mejoró la calidad del agua retenida en el dique de Entremuros subiendo el pH y precipitando los metales pesados. Los mecanismos de entrada de los metales pesados en la cadena trófica parecían ser lentos, dando tiempo a que la retirada de los lodos tóxicos llevada a cabo por la Administración fuera eficaz y diera tiempo a realizar tanto esfuerzo. Aunque el Guadiamar ha trabajado muy duro en su propia recuperación, con su ayuda hemos elaborado una gran cantidad de propuestas técnicas; unas para actuaciones de emergencia, otras a corto, medio y largo plazo. También hemos dado forma a un Plan frente a las previsibles avenidas de este primer otoño después del vertido. Nuestro objetivo ha sido poner a disposición soluciones preparadas para todo tipo de problemas, en primera o en segunda instancia. Prevenir no solo una o dos contingencias, se ha tratado de estar preparado para la mayor cantidad de eventualidades posibles. Por ello algunas serán utilizables, otras estarán en reserva, y muchas irían al cajón de los papeles. Pero ahí están por si acaso. Este libro recoge los trabajos de campo, los ensayos de laboratorio y la ingeniería desarrollada en los primeros cuatro meses. Durante el siguiente preparamos la edición del mismo, mientras, en paralelo, continuábamos en el trabajo experimental y el diseño. Cuando se cumpla el quinto mes, el 25 de Septiembre de 1998, lo presentaremos, ciento cincuenta días después... Con la financiación de la Diputación de Sevilla hemos preparado la primera edición en formato CD Rom e Internet, con muy poco coste para acceder a su contenido. En poco tiempo saldrá la edición en papel, con la misma financiación que la primera. Nos gustaría que este documento fuera entendido como lo que es, en nuestra opinión, una llamada urgente al debate de las ideas. Tratamos de ofrecer la información necesaria y el foro donde recoger las propuestas que seguramente muchos pueden aportar sin saber como transmitir sus experiencias. El Grupo de Tratamiento de Aguas Residuales (T.A.R.) abre con este libro la MESA DE DISCUSIÓN, para buscar un poco de luz, avanzar en las soluciones técnicas a la inmensa tarea de recuperar el río Guadiamar. El libro presenta lagunas, unas por la enorme prisa, otra por falta de datos, muchas por nuestra escasez de conocimientos. Dicen en España que “lo mejor es enemigo de lo bueno”...,y nos gustaría recoger ideas hoy mejor que mañana, que podría ser tarde. Nos comprometemos a seguir trabajando en soluciones técnicas, innovaciones tecnológicas e investigación aplicada a la recuperación del Guadiamar, a conocer lo ocurrido y su remedio. Nos comprometemos a publicar de la misma forma los resultados obtenidos, de manera que la discusión y el debate sigan siempre abiertos. El grupo T.A.R. podría ser un punto de intercambio de conocimientos universal, abierto, respetuoso y tolerante, universitario en definitiva, y por tanto útil en el cumplimiento de sus obligaciones. La primera necesidad de responder urgentemente, está dando paso a unas actuaciones programadas, a medida de los efectos de las correcciones introducidas. Deben instaurarse políticas de prevención y nuevas actuaciones para recuperar el Guadiamar, mejorar urgentemente las condiciones del entorno. Aprender de las soluciones adoptadas y generar mejores prácticas, puede ser una buena conclusión del trabajo realizado por tanta gente. Lo que empezó siendo una carrera de velocidad se nos convierte en un maratón, ya no hay que correr explosivamente, hay que mantener un ritmo en la carrera; hay que persistir en el esfuerzo todos los días durante mucho tiempo. Este nuevo desafío sigue siendo duro y difícil. Podéis contar con el Grupo T.A.R. para recorrer el duro camino de la Recuperación

Unraveling the effect of silent, intronic and missense mutations on VWF splicing : contribution of next generation sequencing in the study of mRNA

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. identifier:02869074

AFLP analysis reveals high genetic diversity but low population structure in Coccidioides posadasiiisolates from Mexico and Argentina

BACKGROUND: Coccidioides immitis and C. posadasii cause coccidioidomycosis, a disease that is endemic to North and South America, but for Central America, the incidence of coccidioidomycosis has not been clearly established. Several studies suggest genetic variability in these fungi; however, little definitive information has been discovered about the variability of Coccidioides fungi in Mexico (MX) and Argentina (AR). Thus, the goals for this work were to study 32 Coccidioides spp. isolates from MX and AR, identify the species of these Coccidioides spp. isolates, analyse their phenotypic variability, examine their genetic variability and investigate the Coccidioides reproductive system and its level of genetic differentiation. METHODS: Coccidioides spp. isolates from MX and AR were taxonomically identified by phylogenetic inference analysis using partial sequences of the Ag2/PRA gene and their phenotypic characteristics analysed. The genetic variability, reproductive system and level of differentiation were estimated using AFLP markers. The level of genetic variability was assessed measuring the percentage of polymorphic loci, number of effective allele, expected heterocygosity and Index of Association (I(A)). The degree of genetic differentiation was determined by AMOVA. Genetic similarities among isolates were estimated using Jaccard index. The UPGMA was used to contsruct the corresponding dendrogram. Finally, a network of haplotypes was built to evaluate the genealogical relationships among AFLP haplotypes. RESULTS: All isolates of Coccidioides spp. from MX and AR were identified as C. posadasii. No phenotypic variability was observed among the C. posadasii isolates from MX and AR. Analyses of genetic diversity and population structure were conducted using AFLP markers. Different estimators of genetic variability indicated that the C. posadasii isolates from MX and AR had high genetic variability. Furthermore, AMOVA, dendrogram and haplotype network showed a small genetic differentiation among the C. posadasii populations analysed from MX and AR. Additionally, the I(A) calculated for the isolates suggested that the species has a recombinant reproductive system. CONCLUSIONS: No phenotypic variability was observed among the C. posadasii isolates from MX and AR. The high genetic variability observed in the isolates from MX and AR and the small genetic differentiation observed among the C. posadasii isolates analysed, suggest that this species could be distributed as a single genetic population in Latin America

Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES) : comprehensive genetic analysis by next-generation sequencing of 480 patients

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population