Hemicraniectomía endonasal por estesioneuroblastoma: informe de un caso / Endonasal hemicraniectomy by esthesioneuroblastoma: report of a case

El estesioneuroblastoma es una neoplasia maligna que se origina del epitelio olfatorio. El tratamiento se establece de acuerdo con su extensión y el grado histológico de atipia y puede incluir cirugía, cirugía más radioterapia o más radioquimioterapia. Se han utilizado diferentes abordajes quirúrgicos que incluyeron incisiones faciales y craneotomía pero, con la mayor experiencia adquirida en cirugía endoscópica de senos paranasales y el trabajo en equipo con el neurocirujano, se han desarrollado técnicas endonasales que posibilitan realizar resecciones oncológicas en pacientes seleccionados, con menos morbilidad, internación breve y sin comprometer el control local de la enfermedad. Describimos el caso clínico de una paciente con un estesioneuroblastoma con invasión intracraneal, que fue tratada con éxito mediante una hemicraniectomía endonasal preservando el bulbo olfatorio contralateral. (AU)

Esthesioneuroblastoma is a malignant neoplasm that originates from the olfactory epithelium. Treatment is established according to its extension and the histological degree of atypia and may include surgery, surgery more radiotherapy or more chemoradiation therapy. Different surgical approaches have been used, including facial incisions and craniotomy, but with the greater experience acquired with endoscopic sinus surgery and teamwork with the neurosurgeon, endonasal techniques have been developed that make it possible to perform oncological resections in selected patients, with less morbidity, brief hospitalization and without compromising local control of the disease. We describe the clinical case of a patient with an esthesioneuroblastoma with intracranial invasion who was successfully treated by endonasal hemicraniectomy preserving the contralateral olfactory bulb. (AU)

The Lack of the TetR-Like Repressor Gene BCG_2177c (Rv2160A) May Help Mycobacteria Overcome Intracellular Redox Stress and Survive Longer Inside Macrophages When Surrounded by a Lipid Environment.

Mycobacteria, like other microorganisms, survive under different environmental variations by expressing an efficient adaptive response, oriented by regulatory elements, such as transcriptional repressors of the TetR family. These repressors in mycobacteria also appear to be related to cholesterol metabolism. In this study, we have evaluated the effect of a fatty acid (oleic-palmitic-stearic)/cholesterol mixture on some phenotypic and genotypic characteristics of a tetR-mutant strain (BCG_2177c mutated gene) of M. bovis BCG, a homologous of Rv2160A of M. tuberculosis. In order to accomplish this, we have analyzed the global gene expression of this strain by RNA-seq and evaluated its neutral-lipid storage capacity and potential to infect macrophages. We have also determined the macrophage response by measuring some pro- and anti-inflammatory cytokine expressions. In comparison with wild-type microorganisms, we showed that the mutation in the BCG_2177c gene did not affect the growth of M. bovis BCG in the presence of lipids but it probably modified the structure/composition of its cell envelope. Compared to with dextrose, an overexpression of the transcriptome of the wild-type and mutant strains was observed when these mycobacteria were cultured in lipids, mainly at the exponential phase. Twelve putative intracellular redox balance maintenance genes and four others coding for putative transcriptional factors (including WhiB6 and three TetR-like) were the main elements repeatedly overexpressed when cultured in the presence of lipids. These genes belonged to the central part of what we called the "genetic lipid signature" for M. bovis BCG. We have also found that all these mycobacteria genotypic changes affected the outcome of BCG-infected macrophages, being the mutant strain most adapted to persist longer inside the host. This high persistence result was also confirmed when mutant-infected macrophages showed overexpression of the anti-inflammatory cytokine TGF-ß versus pro-inflammatory cytokines. In summary, the lack of this TetR-like repressor expression, within a lipid environment, may help mycobacteria overcome intracellular redox stress and survive longer inside their host.

Close Related Drug-Resistance Beijing Isolates of Mycobacterium tuberculosis Reveal a Different Transcriptomic Signature in a Murine Disease Progression Model.

Mycobacterium tuberculosis (MTB) lineage 2/Beijing is associated with high virulence and drug resistance worldwide. In Colombia, the Beijing genotype has circulated since 1997, predominantly on the pacific coast, with the Beijing-Like SIT-190 being more prevalent. This genotype conforms to a drug-resistant cluster and shows a fatal outcome in patients. To better understand virulence determinants, we performed a transcriptomic analysis with a Beijing-Like SIT-190 isolate (BL-323), and Beijing-Classic SIT-1 isolate (BC-391) in progressive tuberculosis (TB) murine model. Bacterial RNA was extracted from mice lungs on days 3, 14, 28, and 60. On average, 0.6% of the total reads mapped against MTB genomes and of those, 90% against coding genes. The strains were independently associated as determined by hierarchical cluster and multidimensional scaling analysis. Gene ontology showed that in strain BL-323 enriched functions were related to host immune response and hypoxia, while proteolysis and protein folding were enriched in the BC-391 strain. Altogether, our results suggested a differential bacterial transcriptional program when evaluating these two closely related strains. The data presented here could potentially impact the control of this emerging, highly virulent, and drug-resistant genotype.

Dissecting industrial fermentations of fine flavour cocoa through metagenomic analysis.

The global demand for fine-flavour cocoa has increased worldwide during the last years. Fine-flavour cocoa offers exceptional quality and unique fruity and floral flavour attributes of high demand by the world's elite chocolatiers. Several studies have highlighted the relevance of cocoa fermentation to produce such attributes. Nevertheless, little is known regarding the microbial interactions and biochemistry that lead to the production of these attributes on farms of industrial relevance, where traditional fermentation methods have been pre-standardized and scaled up. In this study, we have used metagenomic approaches to dissect on-farm industrial fermentations of fine-flavour cocoa. Our results revealed the presence of a shared core of nine dominant microorganisms (i.e. Limosilactobacillus fermentum, Saccharomyces cerevisiae, Pestalotiopsis rhododendri, Acetobacter aceti group, Bacillus subtilis group, Weissella ghanensis group, Lactobacillus_uc, Malassezia restricta and Malassezia globosa) between two farms located at completely different agro-ecological zones. Moreover, a community metabolic model was reconstructed and proposed as a tool to further elucidate the interactions among microorganisms and flavour biochemistry. Our work is the first to reveal a core of microorganisms shared among industrial farms, which is an essential step to process engineering aimed to design starter cultures, reducing fermentation times, and controlling the expression of undesirable phenotypes.

Recovery and functional validation of hidden soil enzymes in metagenomic libraries.

The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.

Hypoxia Is Not a Main Stress When Mycobacterium tuberculosis Is in a Dormancy-Like Long-Chain Fatty Acid Environment.

The capacity of Mycobacterium tuberculosis (Mtb) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, Mtb is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of Mtb to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the in vitro Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that sigB and sigE were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of Mtb to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of Mtb to establish long-term survival during latent infection.

Bioelectrochemical Detection of Mycobacterium tuberculosis ESAT-6 in an Antibody-Based Biomicrosystem.

Bioelectrochemical sensing of Mycobacterium tuberculosis through electro-immunosensors is a promising technique to detect relevant analytes. In general, immunosensors require the formation of organic assemblies by the adsorption of molecular constituents. Moreover, they depend on the correct immobilization of the bio-recognition element in the biosensor. These procedures cannot be easily monitored without the use of invasive methods. In this work, an impedance analysis technique was used, as a non-invasive method, to measure and differentiate the manufacturing stages of the sensors. Biomicrosystems were fabricated through physical vapor deposition (PVD) of 80 nm Au nanolayers on 35 µm copper surfaces. Later, the surface was modified through thiolation methods generating a self-assembled-monolayer (SAM) with 20 mM 4-aminothiophenol (4-ATP) on which a polyclonal antibody (pAb) was covalently attached. Using impedance analysis, every step of the electro-immunosensor fabrication protocol was characterized using 40 independent replicas. Results showed that, compared to the negative controls, distilled water, and 0.5 µg/mL HSA, a maximum variation of 171% between each replica was achieved when compared to samples containing 0.5 µg/mL of ESAT-6 M. tuberculosis immunodominant protein. Therefore, this development validates a non-invasive method to electrically monitor the assembly process of electro-immunosensors and a tool for its further measure for detection of relevant antigens.

Resultados y consideraciones técnicas en la rinoplastia y cierre de perforación septal combinados / Results and technical considerations in combined rhinoplastyand septal perforation repair / Resultados e considerações técnicas na rinoplastia e fechamento de perfuração septal combinados

Introducción: la combinación de dos técnicas como la rinoplastia y el cierre de perforación septal representa un desafío quirúrgico. El objetivo de este trabajo es presentar los resultados a largo plazo y las dificultades en las técnicas quirúrgicas utilizadas al realizar ambos procedimientos en simultáneo. Métodos: Se realizó un estudio descriptivo retrospectivo que incluyó un total de 6 pacientes sometidos a rinoplastia y cierre de perforación septal combinados. Se evaluaron antecedentes personales, motivos estéticos, síntomas, tamaño de la perforación septal, técnica quirúrgica, injertos utilizados y sitio donante. Se analizaron las complicaciones y los resultados subjetivos y objetivos obtenidos. Resultados: La edad promedio de los pacientes fue de 37,5 años. Los motivos de consulta estéticos más frecuentes fueron presencia de giba osteocartilaginosa y combinación de punta nasal ancha e hiporrotada. El síntoma funcional más frecuente fue la obstrucción nasal. En todos los casos se realizó cirugía por abordaje abierto. Se llevó a cabo el cierre de la perforación septal mediante la técnica de colgajo bipediculado con interposición de cartílago envuelto en fascia temporal. El cierre completo de la perforación se logró en 5 pacientes. En todos los casos se logró mejoría sintomática y se cumplió el objetivo estético propuesto. Conclusiones: Realizar una rinoplastia con cierre de perforación septal combinado mediante un correcto plan preoperatorio es un procedimiento viable, desafiante y eficaz en cirujanos entrenados. El abordaje abierto facilita el cierre de la perforación septal sin afectar el resultado estético.

Introduction: the combination of two surgical techniques such as rhinoplasty and septal perforation repair, present a surgical challenge. The objective of this study is to show long-term results and difficulties in the surgical techniques used when performing both procedures simultaneously. Methods: A retrospective descriptive study which included six (6) patients who underwent combined rhinoplasty and repair of septal perforation. Personal history, aesthetic motives, symptoms, size of the septal perforation, surgical techniques, grafts used and donor site we evaluated. The complications and subjective and objective results obtained were analyzed. Results: A total of 6 patients were included with an average age of 37.5 years. The most frequent motives for aesthetic consultation were the presence of a dorsal hump and the combination of a wide nasal tip with reduced tip rotation. The most frequent functional motive was nasal obstruction. An external approach was used in all the cases. The septal repair was performed with bipedicled advancement mucosal flaps and concurrent interposed composite graft formed with cartilage and temporal fascia. Complete repair was achieved in five (5) patients. Symptom improvement and aesthetic goals were reached in all the patients. Conclusions: Combined rhinoplasty and septal perforation repair is a viable, challenging and effective procedure when done by trained surgeons with a correct surgical plan. The external approach facilitates the septal perforation repair and this repair dose not affect the aesthetic outcome.

Introdução: a combinação de dois técnicas cirúrgicas como a Rinoplastia e o fechamento de perfuração septal representa um desafio cirúrgico. O objetivo deste trabalho é apresentar os resultados a longo prazo e as dificuldades nas técnicas cirúrgicas utilizadas ao realizar ambos procedimentos em simultâneo. Métodos: Realizouse um estudo descritivo retrospectivo que incluiu (6) seis pacientes sometidos a rinoplastia e fechamento de perfuração septal combinados. Avaliaram-se antecedentes pessoais, motivos estéticos, sintomas, tamanho da perfuração septal, técnica cirúrgica, enxertos utilizados e sitio doador. Analisaram-se as complicações e os resultados subjetivos e objetivos obtidos. Resultados: Incluíram-se um total de 6 pacientes com uma idade média de 37,5 anos. Os motivos de consulta estéticos mais frequentes foram presença de giba osteocartilaginosa e combinação de ponta nasal larga e hiporrotada. O motivo funcional mais frequente foi a obstrução nasal. Em todos os casos realizou-se uma cirurgia por abordagem aberta. Levou- se a cabo o fechamento da perfuração septal mediante a técnica de retalho bipediculado com interposição de cartilagem envolta em fáscia temporal. O fechamento completo da perfuração logrouse em 5 pacientes. Em todos os pacientes logrou-se melhoria sintomática e cumpriu-se o objetivo estético proposto. Conclusões: Realizar uma rinoplastia com fechamento de perfuração septal combinados é um procedimento viável, desafiante e eficaz em cirurgiões treinados com um correto plano pré-operatório. A abordagem aberta facilita o fechamento da perfuração septal, enquanto as reparações do mesmo não afetam o resultado estético.

Clonación, expresión y caracterización de una nueva esterasa derivada de metagenomas de suelos agrícolas colombianos / Cloning, expression and characterization of a novel esterase derived from colombian agricultural soils metagenomes

El presente trabajo tuvo como objetivo la bioprospección de ADN metagenómico derivado de comunidades microbianas asociadas a un agroecosistema de importancia nacional. Este análisis permitió realizar la producción, expresión, purificación y caracterización de una enzima novedosa con actividad esterasa. Esta enzima, denominada LipM, había sido previamente identificada en clones metagenómicos derivados de suelos dedicados al cultivo de papa criolla (Solanum pureja), mediante secuencia de nueva generación y análisis bioinformáticos. La secuencia codificante de la enzima fue clonada en el vector pBADgiii y expresada en E. coli como sistema de expresión, lo que permitió optimizar el proceso de producción recombinante y su posterior purificación. Funcionalmente la enzima presentó una mayor afinidad por sustratos de p-nitrofenil con ácidos grasos de cadena corta (

The present work had as a main objective to bioprospect metagenomic DNA from microbial communities associated with an agro-ecosystem of national importance. This analysis allowed the production, expression, purification and characterization of a novel enzyme with esterase activity. This enzyme, named here as LipM, was previously identified in metagenomic clones derived from soils dedicated to creole potato (Solanum pureja) crops by means of next-generation sequencing and bioinformatics analyses. The coding sequence of the enzyme was cloned into pBADgiii vector and expressed in E. coli as an expression system, allowing to optimize its recombinant production process and its further purification. The enzyme functionally showed a greater affinity for p-nitrophenyl substrates with short-chain fatty acids (

Development of a genetic tool for functional screening of anti-malarial bioactive extracts in metagenomic libraries.

BACKGROUND: The chemical treatment of Plasmodium falciparum for human infections is losing efficacy each year due to the rise of resistance. One possible strategy to find novel anti-malarial drugs is to access the largest reservoir of genomic biodiversity source on earth present in metagenomes of environmental microbial communities. METHODS: A bioluminescent P. falciparum parasite was used to quickly detect shifts in viability of microcultures grown in 96-well plates. A synthetic gene encoding the Dermaseptin 4 peptide was designed and cloned under tight transcriptional control in a large metagenomic insert context (30 kb) to serve as proof-of-principle for the screening platform. RESULTS: Decrease in parasite viability consistently correlated with bioluminescence emitted from parasite microcultures, after their exposure to bacterial extracts containing a plasmid or fosmid engineered to encode the Dermaseptin 4 anti-malarial peptide. CONCLUSIONS: Here, a new technical platform to access the anti-malarial potential in microbial environmental metagenomes has been developed.

Global adaptation to a lipid environment triggers the dormancy-related phenotype of Mycobacterium tuberculosis.

UNLABELLED: Strong evidence supports the idea that fatty acids rather than carbohydrates are the main energy source of Mycobacterium tuberculosis during infection and latency. Despite that important role, a complete scenario of the bacterium's metabolism when lipids are the main energy source is still lacking. Here we report the development of an in vitro model to analyze adaptation of M. tuberculosis during assimilation of long-chain fatty acids as sole carbon sources. The global lipid transcriptome revealed a shift toward the glyoxylate cycle, the overexpression of main regulators whiB3, dosR, and Rv0081, and the increased expression of several genes related to reductive stress. Our evidence showed that lipid storage seems to be the selected mechanism used by M. tuberculosis to ameliorate the assumed damage of reductive stress and that concomitantly the bacilli acquired a slowed-growth and drug-tolerant phenotype, all characteristics previously associated with the dormant stage. Additionally, intergenic regions were also detected, including the unexpected upregulation of tRNAs that suggest a new role for these molecules in the acquisition of a drug-tolerant phenotype by dormant bacilli. Finally, a set of lipid signature genes for the adaptation process was also identified. This in vitro model represents a suitable condition to illustrate the participation of reductive stress in drugs' activity against dormant bacilli, an aspect scarcely investigated to date. This approach provides a new perspective to the understanding of latent infection and suggests the participation of previously undetected molecules. IMPORTANCE: Mycobacterium tuberculosis establishes long-lasting highly prevalent infection inside the human body, called latent tuberculosis. The known involvement of fatty acids is changing our understanding of that silent infection; however, question of how tubercle bacilli globally adapt to a lipid-enriched environment is still an unanswered. With the single change of providing fatty acids as carbon sources, the bacilli switch on their program related to dormant stage: slowed growth, accumulation of lipid bodies, and development of drug tolerance. In this stage, unexpected and previously unknown participants were found to play putatively important roles during the process. For the first time, this work compares the global transcriptomics of bacteria by using strand-specific RNA sequencing under two different growth conditions. This study suggests novel targets for the control of tuberculosis and provides a new straightforward in vitro model that could help to test the activity of drugs against dormant bacilli from a novel perspective.

Computational modeling and preliminary iroN, fepA, and cirA gene expression in Salmonella Enteritidis under iron-deficiency-induced conditions.

Salmonellosis outbreaks in Europe, the United States, and Latin America have been associated with contaminated food derivatives including meat from the poultry industry. Salmonella grown under iron-limiting conditions has the capability to increase concentration of several iron-regulated outer-membrane proteins to augment the acquisition of the metal. These proteins have been proved to have immunogenic properties. Our aim was to increase the relative expression of iroN, fepA, and cirA in Salmonella Enteritidis domestic strain. Furthermore, we proposed a 3-dimensional structure model for each protein to predict and locate antigenic peptides. Our eventual objective is to produce an effective vaccine against regional avian salmonellosis. Two simple factorial designs were carried out to discriminate between 2 nitrogen sources and determine chelating-agent addition timing to augment relative gene expression. Two antigenic peptides located at the external face of each protein and 2 typical domains of iron-regulated outer-membrane proteins, plug and TonB-dep-Rec, were identified from the 3-dimensional models. Tryptone was selected as the best nitrogen source based on growth rate (µx = 0.36 h(-1)) and biomass productivity (Px = 0.9 g•h(-1)•L(-1)) as determined by a general factorial design. Optimum timing for chelating agent addition was in the middle of the log phase, which allowed relative expressions at 4 h of culture. Increase in iroN, fepA, and cirA relative expression was favored by the length of log phase and the addition of chelating agent, which decreased chelating toxicity and enhanced cell growth rate.

Composition andffunction of the microbial commuity related with the nitrogen ccycling on the potato rhizosphere / Composición y función de la comunidad microbiana relacionada con el ciclaje de nitrógeno en la rizosfera de Solanum tuberosum (BAHUIN) grupo phureja

In the S. tuberosum group phureja crops, mineral fertilizer and organic amendments are applied to meet the plants´ nutritional demands, however the effect of such practices on the associated rizospheric microbial communities are still unknown. Nitrogen plays an important role in agricultural production, and a great diversity of microorganisms regulates its transformation in the soil, affecting its availability for the plant. The aim of this study was to assess the structure of microbial communities related with the N cycle of S. tuberosum group phureja rizospheric soil samples, with contrasting physical-chemical properties and fertilization strategy. Few significant differences between the community composition at the phylum level were found, only Planctomycetes phylum was different between samples of different soil type and fertilization strategy. However, the analysis of nitrogen-associated functional groups made by ribotyping characterization, grouped soils in terms of such variables in a similar way to the physical-chemical properties. Major differences between soil samples were typified by higher percentages of the ribotypes from nitrite oxidation, nitrogen fixation and denitrification on organic amendment soils. Our results suggest that, the dominant rhizosphere microbial composition is very similar between soils, possibly as a result of population´s selection mediated by the rhizosphere effect. However, agricultural management practices in addition to edaphic properties of sampled areas, appear to affect some functional groups associated with the nitrogen cycling, due to differences found on soil´s physicalchemical properties, like the concentration of ammonium that seems to have an effect regulating the distribution and activity of nitrogen related functional groups in the S. tuberosum rhizosphere.

Fertilización mineral y enmiendas orgánicas son aplicadas para satisfacer las demandas nutricionales de los cultivos de S. tuberosum grupo phureja . Sin embargo, el efecto de esas prácticas sobre la comunidad microbiana asociada a la rizósfera aún no se conocen. El nitrógeno juega un papel importante en la producción agrícola y una gran diversidad de microorganismos regulan su transformación en el suelo, afectando su disponibilidad para la planta. El objeto de este estudio fue determinar la composición de la comunidad microbiana de la rizósfera de S. tuberosum grupo phureja , asociada con el ciclo del nitrógeno, en muestras de suelo contrastantes en sus propiedades fisicoquímicas y estrategia de fertilización. Pocas diferencias significativas entre la composición de la comunidad microbiana a nivel de phylum fueron encontradas, Í°nicamente el phylum Planctomycetes fue diferente entre las muestras de suelos con estrategias de fertilización diferentes. Sin embargo, el análisis de grupos funcionales asociados al nitrógeno llevado a cabo por la caracterización de ribotipificación, agrupó los suelos en términos de esas variables en una forma similar a las propiedades fisicoquímicas del suelo. Diferencias mayores entre las muestras de suelo fueron tipificadas por los altos porcentajes de ribotipos asociados a la oxidación de nitrito, fijación de nitrógeno y denitrificación sobre los suelos con enmiendas orgánicas. Nuestros resultados sugieren que la composición microbiana dominante es muy similar entre suelos, posiblemente como resultado de la selección de poblaciones mediada por el efecto rizosférico. Sin embargo, las prácticas del manejo agrícola en conjunto con las propiedades del suelo en las áreas muestreadas, parecen afectar algunos grupos funcionales asociados con el ciclo de nitrógeno, debido a las diferencias encontradas en las propiedades fisicoquímicas del suelo, como la concentración de amonio que parece tener un efecto regulando la distribución y actividad de los grupos funcionales relacionados con el ciclo del nitrógeno en la rizosfera de S. tuberosum.

Rifampin-isoniazid oligonucleotide typing: an alternative format for rapid detection of multidrug-resistant Mycobacterium tuberculosis.

A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources.

Genomic signatures of the haarlem lineage of Mycobacterium tuberculosis: implications of strain genetic variation in drug and vaccine development.

Tuberculosis is the world's leading cause of death due to a single infectious agent, and efforts aimed at its control require a better understanding of host, environmental, and bacterial factors that govern disease outcome. Growing evidence indicates that certain Mycobacterium tuberculosis strains of distinct phylogeographic lineages elicit unique immunopathological events. However, identifying the genetic basis of these phenotypic peculiarities has proven difficult. Here we report the presence of six large sequence polymorphisms which, together with two single-nucleotide changes previously described by our group, consistently differentiate Haarlem strains from the remaining M. tuberculosis lineages. The six newly found Haarlem-specific genetic events are four deletions, which altogether involve more than 13 kb, and two intragenic insertions of the element IS6110. The absence of the genes involved in these polymorphisms could have an important physiological impact on Haarlem strains, i.e., by affecting key genes, such as Rv1354c and cyp121, which have been recently proposed as plausible drug targets. These lineage-specific polymorphisms can serve as genetic markers for the rapid PCR identification of Haarlem strains, providing a useful tool for strain surveillance and molecular epidemiology studies. Strain variability such as that described here underscores the need for the definition of a core set of essential genes in M. tuberculosis that are ubiquitously present in all circulating lineages, as a requirement in the development of effective antituberculosis drugs and vaccines.

Ecocardiograma de esfuerzo para la evaluación de la enfermedad coronaria / Assessment of coronary artery disease by exercise echocardiography

La ecocardiografía de esfuerzo consiste en la adición de la imagen ecocardiográfica a la prueba de esfuerzo convencional. Tiene un perfil de seguridad, costo, rentabilidad diagnóstica y pronóstica y ausencia de radiación óptimos para la evaluación no invasiva de la enfermedad coronaria. Aparte de su valor indudable en casos de ECG basal no interpretable o en pacientes con resultado no concluyente en la prueba de esfuerzo convencional, las guías recientes la establecen como una alternativa diagnóstica y pronóstica a la prueba de esfuerzo convencional en los centros en que existan facilidades para ello. Su sensibilidad puede incrementarse si se entrena en la adquisición de imagen en pico de ejercicio, en lugar de la imagen posejercicio. Las limitaciones de antaño debidas a una deficiente ventana ecocardiográfica se han solventado en gran medida con el uso de la imagen armónica y el contraste para detección de bordes. La nueva tecnología es prometedora. Particularmente la eco 3D podría tener su papel al poder realizar un estudio completo en sólo unos segundos. Por otra parte, el strain bidimensional (speckle) puede medir el movimiento de torsión miocárdico que está abolido en situaciones de isquemia.

Ecocardiograma de esfuerzo para la evaluación de la enfermedad coronaria / Assessment of coronary artery disease by exercise echocardiography

La ecocardiografía de esfuerzo consiste en la adición de la imagen ecocardiográfica a la prueba de esfuerzo convencional. Tiene un perfil de seguridad, costo, rentabilidad diagnóstica y pronóstica y ausencia de radiación óptimos para la evaluación no invasiva de la enfermedad coronaria. Aparte de su valor indudable en casos de ECG basal no interpretable o en pacientes con resultado no concluyente en la prueba de esfuerzo convencional, las guías recientes la establecen como una alternativa diagnóstica y pronóstica a la prueba de esfuerzo convencional en los centros en que existan facilidades para ello. Su sensibilidad puede incrementarse si se entrena en la adquisición de imagen en pico de ejercicio, en lugar de la imagen posejercicio. Las limitaciones de antaño debidas a una deficiente ventana ecocardiográfica se han solventado en gran medida con el uso de la imagen armónica y el contraste para detección de bordes. La nueva tecnología es prometedora. Particularmente la eco 3D podría tener su papel al poder realizar un estudio completo en sólo unos segundos. Por otra parte, el strain bidimensional (speckle) puede medir el movimiento de torsión miocárdico que está abolido en situaciones de isquemia.(AU)

Tumores malignos de cavidad nasal y senos paranasales / Malignant tumors of the nasal cavity and paranasal sinuses

Los tumores malignos de cavidad nasal y senos paranasales son infrecuentes, comprenden menos del 1% de todas las neoplasias malignas y representan el 3% de los tumores originados en cabeza y cuello. Son más comunes en hombres que en mujeres y se diagnostican con más frecuencia entre los 50 a 70 años de edad. Se hizo un estudio retrospectivo sobre 29 pacientes con tumores malignos de cavidad nasal y senos paranasales tratados en un período de 19 años. Se analizaron los resultados de la evaluación otorrinolaringológica, se determinaron los signos y síntomas más frecuentes de consulta, localización e histopatología de los tumores. El tratamiento varió de acuerdo con la ubicación, extensión, e histopatología del tumor, condición clínica y deseo del paciente. Consistió en cirugía, radioterapia, quimioterapia, cirugía más radioterapia posoperatoria, quimioterapia y radioterapia concurrente. La supervivencia libre de enfermedad (SLE) de los 28 pacientes tratados (se excluyó un paciente tratado con radioterapia y respuesta del 80% que no volvió a control) fue de 64.3%, incluyendo un paciente que murió 7 años después del tratamiento. La supervivencia total (SLE + supervivencia con enfermedad) fue del 71.5%.

Malignant tumors of the nasal cavity and paranasalsinuses are uncommon. They account for less than 1%of all malignant neoplasms and 3% of head and necktumors. Malignancies of the sinonasal tract are more frequent in men than in women and are diagnosed more frequently between the ages of 50 and 70. We conducted a retrospective study of 29 patients with malignant tumors of the nasal cavity and paranasal sinuses treated over aperiod of 19 years. The results of the head and neckphysical exam and pathology studies were thoroughly analyzed, determining the most frequent signs andsymptoms on consultation, and the location andhistopathology of the tumors found. The treatment variedaccording to the location, extension and histopathology of the tumors, the clinical condition of the patient and the patient’s wishes. Treatment options varied between surgery, radiotherapy, quimiotherapy, surgery with postoperative radiotherapy, and concurrent quimioradiotherapy. The disease-free survival rate of the 28patients treated was 64.3% including one patient whodied 7 years after treatment. We excluded one patient,treated with radiotherapy with an initial 80% response, who did not come to follow-up. Overall survival was71.5%.

Fusarium species detected in onychomycosis in Colombia.

Fusarium spp. have frequently been isolated from patients with onychomycosis. In Colombia, several studies have shown that Fusarium is the most common non-dermatophyte mould causing onychomycosis and its spread has increased in the past years. In this study, samples were collected in 2003 and 2004 from 137 patients who were diagnosed with onychomycosis caused by Fusarium spp. Three species of Fusarium were identified: Fusarium solani (64.9%), Fusarium oxysporum (32.8%) and Fusarium verticillioides (2.3%). The diseases were more common in women (73%) than in men (27%) and occurred mainly among adults between 31 and 40 years old. The percentage of patients who had received previous treatments was 63.5%. In the last years, new and improved antifungal agents like echinocandins or new triazoles like voriconazole have been developed. For this reason, susceptibility testing using voriconazole was performed, by broth microdilution and disk diffusion. The results showed that F. solani had the highest minimum inhibitory concentration. Using the disk diffusion test, many of the isolates showed variable susceptibility. Genetic diversity of F. oxysporum isolates was determined by random amplified polymorphic DNA. Twenty isolates belonging to different haplotypes were selected for PCR amplification of a region of the gene encoding α-l-arabinofuranosidase B, a specific test to determine if the isolates were F. oxysporum f. sp. dianthi. On the basis of these PCR results, we found that five out of the 20 F. oxysporum isolates corresponded to f. sp. dianthi.

Caracterización molecular de los genes histona H2A y ARNsno-Cl de Trypanosoma rangeli: aplicación en pruebas diagnósticas / Molecular characterization of histone H2A and snoRNA-Cl genes of Trypanosoma rangeli: application in diagnostic tests

La aplicación de la reacción en cadena de la polimerasa (PCR) para detectar e identificar Trypanosoma rangeli y Trypanosoma rangeli presenta a menudo dificultades de interpretación. Así, algunas pruebas generan la amplificación de bandas similares provenientes de uno de los dos parásitos, fragmentos polimórficos de un mismo parásito, o la prevalencia en la detección de T. cruzi en infecciones mixtas. En este estudio se presentan y analizan los trabajos de investigación básica realizados con el objeto de diseñar y estandarizar pruebas de PCR específicas de cada parásito. Los iniciadores TcH2AF/R se diseñaron sobre la base de la región diferencial observada entre las unidades génicas que contienen los genes h2a en estos tripanosomas. Esta pareja de iniciadores amplifican un fragmento de 234 pb específico para T. cruzi (cepas I y II). Los iniciadores TrF/R2 anillan en las regiones intergénicas del fragmento génico de 801 pb codificante para seis transcritos que forman la agrupación ARNsno-Cl en T. rangeli. Estos iniciadores amplifican un fragmento de 620 pb exclusivo de las cepas KP1(-) y KP1(+) de este parásito. La aplicación de estas PCR en vectores infectados y en pacientes con enfermedad de Chagas muestra que ambas pruebas constituyen herramientas útiles para el diagnóstico y la identificación diferencial de estos tripanosomátidos.

The application of polymerase chain reaction (PCR) to detect Trypanosoma rangeli and Trypanosoma rangeli often presents interpretation challenges. For example, some tests yield the amplification of similar bands from either parasite, polymorphic fragments of the same parasite, or present deviation towards T. cruzi in mixed infections. In this study, the basic researching needed for designing and standardizating specific PCR tests for each parasite species PCR are shown and analyzed. The TcH2AF/R primers were designed on the basis of the differential gene region observed between the histone h2a genic units of these parasites. These primers amplify a specific 234 bp fragment in T. cruzi (T. cruzi I and II strains). The TrF/R2 primers anneal to the intergenic regions of an 801 bp gene fragment encoding for six transcripts that conform the snoRNA-Cl cluster in T. rangeli. These primers amplify a fragment of 620 bp exclusively in KP1(-) and KP1(+) strains of the parasite. The application of these PCR tests in infected vectors and in chagasic patients show that both tests constitute useful tools for the diagnosis and differential identification of these Trypanosomatids. Key words: histone, RNA small nucleolar (snoRNA), polymerase chain reaction (PCR), Trypanosoma.