Métodos analíticos para el estudio de Legionella

Assays for Legionella detection in water samples are one of the aspects included in the Spanish legislation on prevention of Legionnaires ́ disease. The frequency of these assays, laboratories that carry out them, and the required actions that derive from them, regarding colony counts, are included in the maintenance plans when Legionella prevention is carried out in water installations (Real Decree 865/2003). The comparison of our legislation with other legislations or recommendations adopted in other countries (United Kingdom, France, Australia, America) allows to know our degree of demand of some parameters. Bacteria culture is the gold standard method for Legionella detection in water samples, and and there are several normalized assays, such as ISO 11731/98 and 2004 or NF T 90-431/2003 (AFNOR). To help the interpretation of the results, Legionella assays should reflect the standard in which they are based and the limit of detection of the method, that should not be over 100 ufc/L. Moreover, laboratories that carry out these assays should be acredited by our national accreditation body (ENAC). In the last years, fast methods have been developed for Legionella detection based on the amplification of chromosomal DNA in water samples by PCR reactions. PCR assays should not be used alone, but it should be a complement of culture assays, when normative actions are implemented in water installations.Los ensayos para la determinación de Legionella en muestras de agua son uno de los aspectos contemplados en la legislación española sobre prevención de legionelosis. La periodicidad de estos ensayos en función del tipo de instalación, los laboratorios que los realizan, y las acciones correctoras que derivan de ellos en función de los recuentos bacterianos son acciones incluidas en los planes de mantenimiento preventivo de las instalaciones consideradas de riesgo (Real Decreto 865/2003). La comparación de nuestra legislación con otras legislaciones o recomendaciones adoptadas en otros paises (Reino Unido, Francia, Australia, América) permite conocer nuestro grado de exigencia en relación a algunos de los parámetros contemplados. El cultivo de la bacteria es el método de referencia para la detección de Legionella en muestras de agua y existen varios ensayos normalizados, como los estándares ISO 11731/98 y 2004 y NF T 90- 431/2003 (AFNOR). Para ayudar a la interpretación de los resultados, los ensayos deben reflejar el estándar en el que se basan y el límite de detección del método, que no debe ser superior a 100 ufc/L. Además, los laboratorios que realizan estos ensayos deben estar acreditados por nuestra entidad de acreditación ENAC. En los últimos años se han desarrollado métodos rápidos de detección de la bacteria basados en la amplificación de ADN cromosómico en muestras de agua mediante reacciones de PCR. El desarrollo científico de estos métodos va por delante del desarrollo reglamentario, y los ensayos de PCR no deben desplazar a los ensayos de cultivo en cumplimiento de las normativas vigentes, sino que deben complementarlo

Legionnaires’ Disease and Travel in Europe

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Brotes de legionelosis notificados a la Red Nacional de Vigilancia Epidemiológica. Años 1999 a 2009

La legionelosis es una enfermedad de origen ambiental que se transmite al ser humano a través de aerosoles de agua contaminada con la bacteria Legionella pneumophila. Se presenta el análisis descriptivo de los resultados de la investigación de los brotes de legionelosis notificados a la Red Nacional de Vigilancia Epidemiológica (RENAVE) durante el periodo 1999 a 2009

Virulence factor rtx in Legionella pneumophila, evidence suggesting it is a modular multifunctional protein

<p>Abstract</p> <p>Background</p> <p>The repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of <it>Legionella pneumophila </it>and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity.</p> <p>Results</p> <p>The comparative analysis of <it>rtx </it>gene among 6 strains of <it>L. pneumophila </it>showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of <it>rtx </it>among the considered strains, the flanking genes are maintained in synteny and similarity.</p> <p>Conclusion</p> <p>In contrast to the extracellular bacteria <it>Vibrio cholerae</it>, in which the <it>rtx </it>gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen <it>L. pneumophila </it>shows a rapid evolution. Changes in the <it>rtx </it>could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences.</p

Legionella feeleii: Ubiquitous Pathogen in the Environment and Causative Agent of Pneumonia

L. feeleii is one of the most frequent Legionella species isolated from natural pools of the central region of Spain. This study aimed to evaluate its ecology and to identify this Legionella species as a respiratory pathogen. A PCR assay for detecting the L. feeleii mip gene was developed to identify it in clinical and environmental samples. Culture and PCR were performed in environmental samples from four drinking water treatment plants (DWTPs). Free L. feeleii was only detected in raw water samples (3.4%), while L. feeleii as an Acanthamoeba endosymbiont was found in 30.7% of raw water, 11.5% of decanter biofilm, and 32% of finished water samples. Therefore, Acanthamoeba spp. plays an essential role in the multiplication, persistence, and spread of Legionella species in the environment. The first case of Legionnaires' disease caused by L. feeleii in Spain is described in this study. The case was diagnosed in an older woman through PCR and sequencing from urine and sputum samples. A respiratory infection could be linked with health care procedures, and the patient presented several risk factors (age, insulin-dependent diabetes, and heart disease). The detection of non-L. pneumophila, such as L. feeleii, is a factor that must be considered when establishing or reviewing measures for the control and prevention of legionellosis.This work was supported by Instituto de Salud Carlos III (PI12/02725) (PI17/01670) (Co-funded by European Regional Development Fund/European Social Fund “A way to make Europe”/“Investing in your future”); and Fundación Universitaria San Pablo CEU (USPCEU-PC07/2013 and USP-PC07/2014). LV was supported by Universidad San Pablo CEU/Spain (FPI Grants). TG was supported by CAPES/Brazil (BEX 9132/13-9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Legionnaires’ Disease Outbreak in Murcia, Spain

An explosive outbreak of Legionnaires’ disease occurred in Murcia, Spain, in July 2001. More than 800 suspected cases were reported; 449 of these cases were confirmed, which made this the world’s largest outbreak of the disease reported to date. Dates of onset for confirmed cases ranged from June 26 to July 19 , with a case-fatality rate of 1%. The epidemic curve and geographic pattern from the 600 completed epidemiologic questionnaires indicated an outdoor point-source exposure in the northern part of the city. A case-control study matching 85 patients living outside the city of Murcia with two controls each was undertaken to identify the outbreak source; the epidemiologic investigation implicated the cooling towers at a city hospital. An environmental isolate from these towers with an identical molecular pattern as the clinical isolates was subsequently identified and supported that epidemiologic conclusion

Determinación de la Capacidad Térmica y el Calor Específico

Memoria ID-0303. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2014-2015

Classification of follicular-patterned thyroid lesions using a minimal set of epigenetic biomarkers

[Objective]: The minimally invasive fine-needle aspiration cytology (FNAC) is the current gold standard for the diagnosis of thyroid nodule malignancy. However, the correct discrimination of follicular neoplasia often requires more invasive diagnostic techniques. The lack of suitable immunohistochemical markers to distinguish between follicular thyroid carcinoma and other types of follicular-derived lesions complicates diagnosis, and despite most of these tumours being surgically resected, only a small number will test positive for malignancy. As such, the development of new orthogonal diagnostic approaches may improve the accuracy of diagnosing thyroid nodules.[Design]: This study includes a retrospective, multi-centre training cohort including 54 fresh-frozen follicular-patterned thyroid samples and two independent, multi-centre validation cohorts of 103 snap-frozen biopsies and 33 FNAC samples, respectively.[Methods]: We performed a genome-wide genetic and epigenetic profiling of 54 fresh-frozen follicular-patterned thyroid samples using exome sequencing and the Illumina Human DNA Methylation EPIC platform. An extensive validation was performed using the bisulfite pyrosequencing technique.[Results]: Using a random forest approach, we developed a three-CpG marker-based diagnostic model that was subsequently validated using bisulfite pyrosequencing experiments. According to the validation cohort, this cost-effective method discriminates between benign and malignant nodules with a sensitivity and specificity of 97 and 88%, respectively (positive predictive value (PPV): 0.85, negative predictive value (NPV): 0.98).[Conclusions]: Our classification system based on a minimal set of epigenetic biomarkers can complement the potential of the diagnostic techniques currently available and would prioritize a considerable number of surgical interventions that are often performed due to uncertain cytology.[Significance statement]: In recent years, there has been a significant increase in the number of people diagnosed with thyroid nodules. The current challenge is their etiological diagnosis to discount malignancy without resorting to thyroidectomy. The method proposed here, based on DNA pyrosequencing assays, has high sensitivity (0.97) and specificity (0.88) for the identification of malignant thyroid nodules. This simple and cost-effective approach can complement expert pathologist evaluation to prioritize the classification of difficult-to-diagnose follicular-patterned thyroid lesions and track tumor evolution, including real-time monitoring of treatment efficacy, thereby stimulating adherence to health promotion programs.Peer reviewe