The Human Mycobiome in Chronic Respiratory Diseases: Current Situation and Future Perspectives.

Microbes play an important role in the pathogenesis of chronic lung diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, non-cystic fibrosis bronchiectasis, and asthma. While the role of bacterial pathogens has been extensively studied, the contribution of fungal species to the pathogenesis of chronic lung diseases is much less understood. The recent introduction of next-generation sequencing techniques has revealed the existence of complex microbial lung communities in healthy individuals and patients with chronic respiratory disorders, with fungi being an important part of these communities' structure (mycobiome). There is growing evidence that the components of the lung mycobiome influence the clinical course of chronic respiratory diseases, not only by direct pathogenesis but also by interacting with bacterial species and with the host's physiology. In this article, we review the current knowledge on the role of fungi in chronic respiratory diseases, which was obtained by conventional culture and next-generation sequencing, highlighting the limitations of both techniques and exploring future research areas.

Simple method to generate calibrated synthetic smoke-like atmospheres at microscopic scale.

Artificial smokes focusing on macroscopic or fluid properties of smoke have been available for a long time. This paper presents a simple method to generate fully customizable smoke-like atmospheres at microscopic scale (i.e. considering their constituent particles as discrete elements) using a different approach. Synthetic, reproducible media can be generated combining monodisperse microspheres with known geometrical and optical properties conveniently parameterized. The method is presented as a proof-of-concept, highlighting the design decisions along with their implications. Practical issues such as aerosol nebulization, particle carrier selection or the features of the medium chamber where the smoke-like atmosphere is to be tested are analyzed. A comparison between methanol and ethanol as carriers for polystyrene microsphere nebulization is also made. The method could be the seed for the obtention of standard reference media for calibration or standardized characterization of not only smoke detectors and exhaust smoke sensors but also other instruments relying on optical properties of dispersive media (dust in PV panels, public lighting, etc.).

Fungi in Bronchiectasis: A Concise Review.

Although the spectrum of fungal pathology has been studied extensively in immunosuppressed patients, little is known about the epidemiology, risk factors, and management of fungal infections in chronic pulmonary diseases like bronchiectasis. In bronchiectasis patients, deteriorated mucociliary clearance-generally due to prior colonization by bacterial pathogens-and thick mucosity propitiate, the persistence of fungal spores in the respiratory tract. The most prevalent fungi in these patients are Candida albicans and Aspergillus fumigatus; these are almost always isolated with bacterial pathogens like Haemophillus influenzae and Pseudomonas aeruginosa, making very difficult to define their clinical significance. Analysis of the mycobiome enables us to detect a greater diversity of microorganisms than with conventional cultures. The results have shown a reduced fungal diversity in most chronic respiratory diseases, and that this finding correlates with poorer lung function. Increased knowledge of both the mycobiome and the complex interactions between the fungal, viral, and bacterial microbiota, including mycobacteria, will further our understanding of the mycobiome's relationship with the pathogeny of bronchiectasis and the development of innovative therapies to combat it.

Impacto económico de los métodos de diagnóstico rápido en Microbiología Clínica: precio de la prueba o impacto clínico global / Economic impact of rapid diagnostic methods in Clinical Microbiology: Price of the test or overall clinical impact

La necesidad de reducir el tiempo de diagnóstico microbiológico y la irrupción de nuevas tecnologías relacionadas con la microbiología molecular y la proteómica han favorecido el desarrollo de técnicas rápidas y de realización en el lugar de asistencia al paciente (point-of-care), así como de los denominados laboratorios point-of-care, espacios que concentran la realización de ambas técnicas como respuesta, en parte, a la externalización de los laboratorios convencionales de los hospitales. Su introducción no siempre se ha acompañado de evaluaciones económicas (estudios de coste-efectividad, coste-beneficio y coste-utilidad) y suelen limitarse al precio unitario de la prueba. Este último, influido por el procedimiento de compra, no suele tener un valor de referencia regulado, como en el caso de los medicamentos. Los análisis de coste-efectividad que mayor repercusión han tenido han sido los realizados recientemente con la espectrometría de masas en el diagnóstico de la bacteriemia y el uso de antimicrobianos y pueden servir como modelo de futuros estudios económicos de las pruebas rápidas y point-of-care (AU)

The need to reduce the time it takes to establish a microbiological diagnosis and the emergence of new molecular microbiology and proteomic technologies has fuelled the development of rapid and point-of-care techniques, as well as the so-called point-of-care laboratories. These laboratories are responsible for conducting both techniques partially to response to the outsourcing of the conventional hospital laboratories. Their introduction has not always been accompanied with economic studies that address their cost-effectiveness, cost-benefit and cost-utility, but rather tend to be limited to the unit price of the test. The latter, influenced by the purchase procedure, does not usually have a regulated reference value in the same way that medicines do. The cost-effectiveness studies that have recently been conducted on mass spectrometry in the diagnosis of bacteraemia and the use of antimicrobials have had the greatest clinical impact and may act as a model for future economic studies on rapid and point-of-care tests (AU)

Economic impact of rapid diagnostic methods in Clinical Microbiology: Price of the test or overall clinical impact. / Impacto económico de los métodos de diagnóstico rápido en Microbiología Clínica: precio de la prueba o impacto clínico global.

The need to reduce the time it takes to establish a microbiological diagnosis and the emergence of new molecular microbiology and proteomic technologies has fuelled the development of rapid and point-of-care techniques, as well as the so-called point-of-care laboratories. These laboratories are responsible for conducting both techniques partially to response to the outsourcing of the conventional hospital laboratories. Their introduction has not always been accompanied with economic studies that address their cost-effectiveness, cost-benefit and cost-utility, but rather tend to be limited to the unit price of the test. The latter, influenced by the purchase procedure, does not usually have a regulated reference value in the same way that medicines do. The cost-effectiveness studies that have recently been conducted on mass spectrometry in the diagnosis of bacteraemia and the use of antimicrobials have had the greatest clinical impact and may act as a model for future economic studies on rapid and point-of-care tests.

Fluconazole-containing agar Sabouraud dextrose plates are not useful when screening for susceptibility in Candida albicans / Las placas de agar dextrosado de Sabouraud con fluconazol no son útiles para la detección de resistencia en Candida albicans

Introduction. Fluconazole is an alternative for candidemic patients who are not critically ill. Fluconazole is mainly fungistatic and does not completely inhibit visual Candida albicans growth. We studied the usefulness of fluconazolecontaining Sabouraud dextrose agar plates for detecting susceptibility to fluconazole in C. albicans. Material and methods. Adjusted inocula of 19 isolates were transferred directly onto fluconazole-containing Sabouraud dextrose plates (concentrations ranging from 0.125 mg/L to 128 mg/L). The fluconazole MIC in fresh isolates and after growth on the fluconazole-containing plate at 128 mg/L was recorded following the EUCAST EDef 7.2 guidelines. Then isolates were classified according to their degree of trailing production, based on microdilution procedure. Results. All isolates were able to grow on all fluconazolecontaining plates, even those isolates susceptible to fluconazole. In fact, we selected isolates with different degrees of trailing based on microdilution procedures. 50% of isolates classified as heavy trailers, 35.71% as moderate trailers, and 14.28% as slight trailers. Conclusions. The use of fluconazole-containing Sabouraud dextrose agar plates was not a reliable method to detect fluconazole susceptibility in C. albicans isolates; growth of the isolates was a trailing effect rather than true resistance (AU)

Introducción. Fluconazol es el antifúngico de elección en pacientes con candidemia que no están en estado crítico. Fluconazol es un fármaco fungistático y no inhibe por completo el crecimiento de Candida albicans. En este estudio se evalúa la posibilidad de emplear placas de agar dextrosado de Sabouraud con diferentes concentraciones de fluconazol como método para evaluar la sensibilidad a este fármaco de aislados de C. albicans. Material y métodos. El inóculo ajustado procedente de 19 aislados de C. albicans se transfirió directamente a placas que contenían concentraciones de fluconazol comprendidas entre 0,125 mg/L y 128 mg/L. La CMI de fluconazol se calculó en los aislados originales y en los aislados crecidos en la placa de fluconazol de 128 mg/L, según el protocolo de EUCAST EDef 7.2. Los aislados se clasificaron según su grado de producción de efecto de arrastre, siguiendo el procedimiento de microdilución. Resultados. Todos los aislados fueron capaces de crecer en todas las placas con diferentes concentraciones de fluconazol, incluso las cepas sensibles. Los aislados mostraron diferentes grados de efecto de arrastre. El 50% de los aislados se clasificaron como altamente productores, el 35,71% como moderadamente productores y el 14,28% como poco productores de efecto de arrastre. Conclusión. El uso de placas de agar dextrosado de Sabouraud con fluconazol no demostró ser un método útil para el estudio de la sensibilidad de C. albicans a este fármaco ya que el crecimiento de los aislados fue interpretado como efecto de arrastre y no como una verdadera resistencia (AU)

Estudios de coste-efectividad con MALDI-TOF e impacto clínico / Studies of the cost effectiveness of MALDI-TOF and clinical impact

Las nuevas tecnologías suponen en general un incremento del coste económico en los laboratorios por la inversión inicial necesaria para su introducción. Sin embargo, como ha ocurrido con la técnica de espectrometría de masas MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight), con posterioridad se compensa por el desplazamiento de otras técnicas y los beneficios en el paciente derivados de la información nueva generada. Con el uso de MALDI-TOF, de forma directa en el laboratorio de microbiología, se reducen los tiempos de identificación (al menos 24 h) y se mejora el flujo de trabajo, permitiendo una emisión más rápida de los resultados. Este efecto beneficioso ha sido sobre todo estudiado con los frascos de hemocultivos en pacientes con bacteriemia. En ellos, el impacto más analizado ha sido la reducción de la estancia hospitalaria (1,6-6,6 días, dependiendo del tipo de paciente y unidad) y la adecuación del tratamiento. En ambos casos redunda en un ahorro del coste total hospitalario por paciente que puede llegar hasta el 43%. También se ha analizado la reducción de la mortalidad por un mejor manejo precoz de la antibioterapia. En un futuro serán necesarios estudios multicéntricos en los que se incluyan también como factores de análisis aquellos que conllevan cambios en los procesos de información y de actuación clínica acordes con el esfuerzo realizado en el laboratorio de microbiología

In general, new technologies usually increase laboratory costs due to the need for an initial investment. However, as occurred with MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry, this increase is subsequently offset by the discontinued use of traditional technologies and by the benefits to patients of the new information generated. In the clinical microbiology laboratory, the identification time is reduced with the use of MALDI-TOF (by at least 24 hours) and turnaround is improved, allowing faster production of the microbiological report. This beneficial effect has mainly been studied with blood cultures in patients with bacteraemia. In these patients, the length of hospital stay has been reduced by 1.6-6.6 days, depending on the type of patient and the appropriateness of treatment. This leads to better antimicrobial use and a reduction in total hospital cost of up to 43% per patient. Another factor that has been analysed is the decrease in mortality due to better management of antimicrobial therapy. Future multicentre studies should include other factors such as hospital organisation changes and clinical activity arising in response to the efforts of the clinical microbiology laboratory to rapidly obtain information of clinical value

Seguridad en el laboratorio de Microbiología Clínica / Safety in the Microbiology laboratory

La actividad de un laboratorio de Microbiología origina riesgos de distinto tipo, especialmente biológicos, que pueden afectar a la salud de sus trabajadores, de los visitantes y a la comunidad. Los exámenes de salud (vigilancia y prevención), la concienciación individual de autoprotección, la identificación de los peligros y la evaluación del riesgo, la adopción de medidas de contención apropiadas y el empleo de técnicas microbiológicas escrupulosas permiten hacer del laboratorio un lugar seguro, como así demuestran las estadísticas de accidentes o de infecciones contraídas en el laboratorio. Formación e información son las herramientas clave para elaborar un plan de seguridad completo para el laboratorio. En este artículo se revisan los fundamentos teóricos y los conceptos básicos de la seguridad, incluyendo normativas legales. Además se elaboran directrices de tipo práctico para que cada laboratorio planifique su propia seguridad en función de sus características y de su idiosincrasia

The normal activity in the laboratory of microbiology poses different risks - mainly biological - that can affect the health of their workers, visitors and the community. Routine health examinations (surveillance and prevention), individual awareness of self-protection, hazard identification and risk assessment of laboratory procedures, the adoption of appropriate containment measures, and the use of conscientious microbiological techniques allow laboratory to be a safe place, as records of laboratory-acquired infections and accidents show. Training and information are the cornerstones for designing a comprehensive safety plan for the laboratory. In this article, the basic concepts and the theoretical background on laboratory safety are reviewed, including the main legal regulations. Moreover, practical guidelines are presented for each laboratory to design its own safety plan according its own particular characteristics

Microbiological diagnostic procedures for respiratory cystic fibrosis samples in Spain: towards standard of care practices.

BACKGROUND: The microbiological procedures for cystic fibrosis (CF) samples of 17 participating Spanish centers were examined to verify their compliance with current international and national guidelines and to implement the best standards of care for microbiology practices. A 47-item questionnaire covering different CF microbiology aspects was sent to participant laboratories. Telephone interviews were performed when necessary. Data about samples processing for bacteria, mycobacteria and fungi were collected. RESULTS: Gene sequencing (71%), MALDI-TOF (59%) or both (94%) were available for most laboratories. Susceptibility testing was performed by automated microdilution systems (94%) and manual diffusion methods (59%). However, a low use of selective media for Staphylococcus aureus (59%) and Burkholderia cepacia complex (71%), and of epidemiological typing methods (41%) was reported. CONCLUSIONS: Most Spanish laboratories are in agreement with consensus guidelines for the processing of CF respiratory samples, but need to improve in the use of specific selective media and typing methods for epidemiologic studies.

Epidemiology, species distribution and in vitro antifungal susceptibility of fungaemia in a Spanish multicentre prospective survey.

OBJECTIVES: To update the knowledge of the epidemiology of fungaemia episodes in Spain, the species implicated and their in vitro antifungal susceptibilities. METHODS: Episodes were identified prospectively over 13 months at 44 hospitals. Molecular methods were used to determine the cryptic species inside the Candida parapsilosis and Candida glabrata complexes. Susceptibility to amphotericin B, anidulafungin, caspofungin, fluconazole, flucytosine, itraconazole, micafungin, posaconazole and voriconazole was determined by a microdilution colorimetric method. New species-specific clinical breakpoints (SSCBPs) for echinocandins, fluconazole and voriconazole were applied. RESULTS: The incidence of the 1357 fungaemia episodes evaluated was 0.92 per 1000 admissions. The incidence of Candida albicans fungaemia was the highest (0.41 episodes/1000 admissions), followed by Candida parapsilosis sensu stricto (0.22). Candida orthopsilosis was the fifth cause of fungaemia (0.02), outnumbered by Candida glabrata and Candida tropicalis. Interestingly, the incidence of fungaemia by C. parapsilosis was 11 and 74 times higher than that by C. orthopsilosis and Candida metapsilosis, respectively. Neither Candida nivariensis nor Candida bracarensis was isolated. Fungaemia was more common in non-intensive care unit settings (65.2%) and among elderly patients (46.4%), mixed fungaemia being incidental (1.5%). Overall susceptibility rates were 77.6% for itraconazole, 91.9% for fluconazole and 96.5%-99.8% for the other agents. Important resistance rates were only observed in C. glabrata for itraconazole (24.1%) and posaconazole (14.5%), and in Candida krusei for itraconazole (81.5%). CONCLUSIONS: Fungaemia is more common in non-critical patients. C. albicans is the most common species, followed by C. parapsilosis and C. glabrata. Nearly 90% of yeasts are susceptible to all antifungal agents tested. Resistance rates change moderately when applying the new SSCBPs.

Infección bronquial crónica: el problema de Pseudomonas aeruginosa / Chronic bronchial infection: the problem of Pseudomonas aeruginosa

La colonización patogénica broncopulmonar y las exacerbaciones que se derivan de ella constituyen las causasmás importantes del deterioro de la función pulmonar en los pacientes con bronquiectasias. Haemophilusinfluezae y Pseudomonas aeruginosa son los patógenos más frecuentes en estos pacientes. El efecto lesivo seproduce por el proceso de inflamación local y el círculo vicioso que se desarrolla por el estímulo antigénico, laliberación de mediadores de la inflamación, la presencia de neutrófilos, el aumento del inóculo bacteriano yla liberación de exoproductos bacterianos. Se ha demostrado que P. aeruginosa afecta a los pacientes con bronquiectasiascon peor calidad de vida, coloniza a los que tienen peor funcionalidad pulmonar y mayor númerode tratamientos antimicrobianos. En las bronquiectasias, al igual que en la enfermedad pulmonar obstructivacrónica (EPOC) o fibrosis quística, P. aeruginosa es capaz de colonizar crónicamente la mucosa respiratoria.Debido al nicho ecológico donde se sitúa P. aeruginosa y a la multitud de ciclos con antimicrobianos a los queson sometidos estos pacientes es fácil que se desarrollen resistencias a los antimicrobianos, favorecidas por laelevada proporción de variantes hipermutadoras que existen. Asimismo, hay que resaltar la forma natural decrecimiento en biopelículas de P. aeruginosa en la superficie mucosa y la contribución que ejerce para su persistencia.El tratamiento antimicrobiano en los pacientes con bronquiectasas con colonización por P.aeruginosa ha de basarse en antimicrobianos o asociaciones de éstos que no pierdan actividad al actuar sobrelas biopelículas(AU)

Pathogenic bronchopulmonary colonizations and the exacerbations produced are among the most importantcauses of reduced pulmonary function in patients with bronchiectasis. The most frequent pathogens in thesepatients are Haemophilus influenzae and Pseudomonas aeruginosa. Lesions are produced by the localinflammatory process and the vicious circle developed by antigen stimulation, the release of inflammatorymediators, the presence of neutrophils, the increase of bacterial inoculum and the release of bacterialexoproducts. P. aeruginosa has been demonstrated to affect the patients with bronchiectasis and poorestquality of life and to colonize those with the poorest pulmonary function and the highest number ofantimicrobial treatments. In bronchiectasis, as in chronic obstructive pulmonary disease (COPD) or cysticfibrosis, P. aeruginosa is able to colonize the respiratory mucosa chronically. Due to the ecological nicheoccupied by P. aeruginosa and the multitude of cycles with antimicrobial agents to which these patients aresubjected, the development of antimicrobial resistance is highly likely, encouraged by the high proportion ofhypermutation variants in existence. Likewise, P. aeruginosa naturally grows in the form of biofilms on themucosal surface, greatly contributing to its persistence. Antimicrobial treatment in patients withbronchiectasis and P. aeruginosa colonization should be based on antimicrobial agents, alone or incombination, that do not lose activity when acting on biofilms(AU)