Vigilancia epidemiológica de la legionelosis en España: 2005-2015 / Epidemiological surveillance of legionellosis in Spain: 2005-2015

Introducción: La legionelosis es una enfermedad de declaración obligatoria en España. Uno de sus mecanismos de prevención y control es el sistema de vigilancia epidemiológica y, en particular, la investigación epidemiológica. Entre 2005 y 2010 se reportó en Europa un aumento de la carga de la enfermedad no descrita en España. Objetivo: Determinar la evolución de los casos de legionelosis en España en la serie 2005-2015. Métodos: Estudio descriptivo de series temporales relativo al recuento de casos notificados a través del Centro Nacional de Epidemiología de España. Se incluyeron variables sociodemográficas del paciente, antecedentes personales y clínicos, síntomas y signos, datos de laboratorio y epidemiológicos. Se determinó la tasa de incidencia por 100 000 habitantes (2005-2010) y la tasa ajustada (población europea) por 100 000 habitantes según sexo (2005-2015), así como según grupo de edad y sexo para la serie 2010-2015. Resultados: España mantiene una tendencia estable respecto a la tasa de incidencia por 1100 000 habitantes (3,5 a 2,5), se produce un incremento relevante en la tasa ajustada a partir de los 50 años, con mayor impacto en los hombres. Conclusión: se evidencia la necesidad de la vigilancia epidemiológica de la legionelosis, la mejora en las medidas de prevención y control, y la consideración de nuevos factores de riesgo(AU)

Introduction: Legionellosis is a notifiable disease in Spain. One of its prevention and control mechanisms is epidemiological surveillance, particularly epidemiological research. An increase in legionellosis disease burden was reported in Europe from 2005 to 2010 which was not described in Spain. Objectives: Determine the evolution of legionellosis cases in Spain in the period 2005-2015. Methods: A descriptive time series analysis was performed based on the cases notified to the Spanish National Epidemiology Center. The variables considered were the patients' sociodemographic characteristics, personal and clinical antecedents, signs and symptoms, laboratory results and epidemiological data. Determination was made of the incidence rate per 100 000 inhabitants (2005-2010) and the adjusted rate per 100 000 inhabitants (European population) by sex (2005-2015) and by age group and sex for the series (2010-2015). Results: Incidence per 100 000 inhabitants has remained stable (3.5 to 2.5) in Spain, which has led to a relevant increase in the adjusted rate as of age 50 years, with a higher impact among men. Conclusion: Evidence was found of the need for epidemiological surveillance of legionellosis, improvement of prevention and control measures, and consideration of new risk factors(AU)

Legionellosis after hematopoietic stem cell transplantation.

Limited data are available on legionellosis after hematopoietic stem cell transplant (HSCT). The aim of this study was to report the cases of legionellosis and to identify predictors of legionellosis, legionellosis-associated death, and non-relapse mortality (NRM). All cases of post-HSCT legionellosis from the EBMT registry were included and matched with controls in a 3:1 ratio for the analyses of risk factors. In the years 1995-2016, 80 cases from 52 centers in 14 countries were identified (mainly from France, Italy, and Spain). Median time from HSCT to legionellosis was 203 days (range, 0-4099); 19 (23.8%) patients developed early legionellosis (within-day +30 post-HSCT). Patients were mainly male (70%), after allogeneic HSCT (70%), with acute leukemia (27.5%), lymphoma (23.8%), or multiple myeloma (21.3%), and the median age of 46.6 (range, 7.2-68.2). Predictors of legionellosis were allogeneic HSCT (OR = 2.27, 95%CI:1.08-4.80, p = 0.03) and recent other infection (OR = 2.96, 95%CI:1.34-6.52, p = 0.007). Twenty-seven (33.8%) patients died due to legionellosis (44% after early legionellosis), NRM was 50%. Predictors of NRM were female sex (HR = 2.19, 95%CI:1.13-4.23, p = 0.02), early legionellosis (HR = 2.24, 95%CI:1.13-4.46, p = 0.02), and south-eastern geographical region (HR = 2.16, 95%CI:1.05-4.44, p = 0.036). In conclusion, legionellosis is a rare complication after HSCT, mainly allogeneic, occurring frequently within 30 days after HSCT and associated with high mortality.

Mortalidad por neumonía y legionelosis en España: un estudio de series temporales / Pneumonia and legionellosis mortality in Spain: a time series study

Background: Pneumonia is a disease with great relevance in public health, as a leading individual cause of infant mortality worldwide. Legionellosis is a respiratory disease with a bacterial origin and two different clinical forms. Aim: To determine pneumonia and legionellosis mortality in Spain. Material and Methods: Time series study of pneumonia and legionellosis in Spain in two periods, from 1997 to 2001 and from 2011 to 2015. Mortality was calculated according to disease and sex, number of deaths and rates per 100,000 inhabitants. Results: Pneumonia mortality in the first period shows a relatively stable and similar tendency according to sex, preferably affecting males. In the second period, pneumonia mortality increased significantly in recent years. Although pneumonia mortality in Spain decreased in both sexes in some age groups (especially 75 years. Deaths due to legionellosis were relevant in 1997, 1998 and 2000 and increased in the last period. Conclusions: A higher mortality due to pneumonia along the years was identified. Strategies to reduce the incidence and improve the diagnosis of pneumonia, especially in children and older people elderly, are encouraged.

Legionelosis en España, 2010-2015 / Legionellosis in Spain, 2010-2015

Resumen | Introducción. La legionelosis es una enfermedad respiratoria bacteriana de origen ambiental que puede ser adquirida en el ámbito comunitario u hospitalario, y suele estar asociada con equipos, instalaciones y edificios. La forma clínica más conocida es la neumónica, conocida como enfermedad del legionario. Objetivo. Determinar la evolución de los casos de legionelosis en España en el periodo de 2010 a 2015. Materiales y métodos. Se hizo un estudio descriptivo de series temporales y se analizaron los casos de legionelosis notificados al Centro Nacional de Epidemiología del Gobierno de España. Se determinó la distribución de los casos según el sexo, la comunidad autónoma, el mes y los grupos de edad. Para el último se diferenció entre hombres y mujeres. Resultados. El recuento de casos en hombres fue superior al doble con respecto a las mujeres. La distribución en las comunidades autónomas presentó un aumento de los casos notificados al final del periodo en nueve de ellas, siendo notable en Castilla y León, Navarra y el País Vasco, y muy relevante en Castilla-La Mancha. Se estableció un patrón estacional con un pico epidémico en julio-septiembre y un mayor número de casos en torno a los 50 años de edad en ambos sexos. Conclusiones. A pesar de mostrar una prevalencia baja con respecto a otras enfermedades respiratorias, la legionelosis tiene gran impacto en la salud pública. Presenta una distribución global y heterogénea en el territorio español, con un aumento de casos en los dos últimos años, por lo que se requiere una mejor prevención y control de la enfermedad.

Abstract | Introduction: Legionellosis is a bacterial respiratory disease with an environmental origin in the community or in hospitals; it is usually associated with devices, facilities, and buildings. The most common clinical form is the pneumonic, known as legionnaires' disease. Objective: To determine the evolution of legionellosis cases in Spain from 2010 to 2015. Materials and methods: This was a descriptive study of time series with an analysis of the legionellosis cases notified to the Centro Nacional de Epidemiología (Government of Spain). Case distribution was determined according to sex, the autonomous community of origin, month, and age groups differentiating in the latter between men and women. Results: Case count in men was more than double compared to that in women. The cases notified by nine of the autonomous communities showed an increase at the end of the period, especially in Castilla y León, Navarra, and the Basque Country but also notable in Castilla-La Mancha. A seasonal pattern was identified with an epidemic peak in July-September and a greater number of cases among 50-years old people from both sexes. Conclusion: Despite its low prevalence compared to other respiratory diseases, legionellosis has a great impact on public health. Its distribution in Spain is global and heterogeneous with cases increasing in the last two years. Therefore, better disease prevention and control measures are recommended.

Disseminated Legionella micdadei infection in a liver transplant patient presenting as pulmonary nodules and a laryngeal lesion.

We report a liver transplant patient with disseminated Legionella micdadei infection with pulmonary, laryngeal, and suspected muscle involvement. This organism, which stains weakly acid-fast, primarily affects immunocompromised patients. The diagnosis is difficult to make; in this case, the organism was identified via molecular diagnostics on laryngeal and pulmonary biopsy tissue.

Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection.

Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host-pathogen interactions.

Cutaneous Legionella infections in allogeneic hematopoietic cell transplantation recipients.

To date, only twenty cases of cutaneous legionellosis have been reported. Cutaneous legionellosis has heterogeneous manifestations including abscesses, nodules, and cellulitis. The detection of most cutaneous Legionella species requires specific diagnostic cultures and assays. Herein, we report a case of cutaneous legionella in a hematopoietic cell transplantation recipient with culture-negative nodules unresponsive to empiric antibiotics. We also discuss the varied morphology of cutaneous legionellosis and important diagnostic considerations.

Quorum sensing modulates the formation of virulent Legionella persisters within infected cells.

The facultative intracellular bacterium Legionella pneumophila replicates in environmental amoebae and in lung macrophages, and causes Legionnaires' disease. Here we show that L. pneumophila reversibly forms replicating and nonreplicating subpopulations of similar size within amoebae. The nonreplicating bacteria are viable and metabolically active, display increased antibiotic tolerance and a distinct proteome, and show high virulence as well as the capacity to form a degradation-resistant compartment. Upon infection of naïve or interferon-γ-activated macrophages, the nonreplicating subpopulation comprises ca. 10% or 50%, respectively, of the total intracellular bacteria; hence, the nonreplicating subpopulation is of similar size in amoebae and activated macrophages. The numbers of nonreplicating bacteria within amoebae are reduced in the absence of the autoinducer synthase LqsA or other components of the Lqs quorum-sensing system. Our results indicate that virulent, antibiotic-tolerant subpopulations of L. pneumophila are formed during infection of evolutionarily distant phagocytes, in a process controlled by the Lqs system.

[Legionella spp: An update]. / Actualités sur les infections à Legionella.

Legionella-related disease is caused by an intracellular bacteria mainly living in water. Contamination results from inhalation of Legionella sp containing aerosolized water. Main risk factors are tobacco, immunodeficiency, and advanced age. Antigenuria is the cornerstone of the diagnosis. Immunocompromised patients, more commonly infected with non pneumophilaLegionella, present negative antigenuria, and culture and PCR are essential for the diagnosis. Legionnaires' disease may be severe, especially in elderly and/or immunocompromised patients. Mortality rate varies from 10 % in the general population to 50 % in intensive care. Treatment is based on macrolides or fluoroquinolones. Antibiotic resistance is very rare.

Legionelosis ocupacional en mayores de 18 años: revisión sistemática / Occupational legionella in adults over 18 years pf age: a systematic review

Resumen El objetivo es revisar la literatura científica sobre los problemas de salud derivados de la exposición laboral a Legionella spp. Revisión sistemática de la literatura científica recogida en las bases de datos MEDLINE (Pubmed), ISI-Web of Science (Institute for Scientific Information), Cochrane Library Plus, Literatura Latinoamericana de Información en Ciencias de la Salud (LILACS) y SCOPUS, hasta febrero 2015. Los descriptores utilizados fueron: "Legionnaires' Disease" "Legionellosis" "Occupational Exposure" "Occupational Diseases". Se obtuvieron 222 referencias, que tras aplicar los criterios de inclusión y exclusión, se seleccionaron 13 artículos a texto completo. De ellos 9 artículos describen la aparición de neumonía, 4 Fiebre de Pontiac, 1 legionelosis o presunta legionelosis y 3 muerte. El principal agente causal en los profesionales expuestos fue Legionella pneumophila serogrupo 1. La infección por legionela está ligada a las profesiones donde existe nebulización por agua, principalmente en los trabajadores sometidos a largas exposiciones o incluso a la re-exposición. Los brotes se producen de forma estacional, sobre todo en los meses más cálidos.

Abstract The scope of this paper is to review the available scientific literature about the effects on health of occupational exposure to Legionella spp. A systematic review of the scientific literature retrieved from the MEDLINE (via PubMed), ISI-Web of Science (Institute for Scientific Information), Cochrane Library, LILCAS and SCOPUS databases through February 2015 was conducted. The key words used were ««Legionnaires' Disease¼ «Legionellosis¼ «Occupational Exposure¼ «Occupational Diseases¼. Two hundred and twenty-two references were retrieved of which, after applying inclusion/exclusion criteria, 13 complete articles were selected. Of these, 9 describe pneumonia, 4 list Pontiac Fever, 1 reveals legionellosis and 3 result in death. The main causative agent of disease in workers exposed was Legionella pneumophila serogroup 1. Legionella spp infection is closely related to professions where there is nebulization with water, mainly among workers subjected to long exposure or even re-exposure. Outbreaks occurs seasonally, especially in the hottest months.

PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.

The Pathometabolism of Legionella Studied by Isotopologue Profiling.

Metabolic pathways and fluxes can be analyzed under in vivo conditions by incorporation experiments using general 13C-labelled precursors. On the basis of the isotopologue compositions in amino acids or other metabolites, the incorporation rates of the supplied precursors and the pathways of their utilization can be studied in considerable detail. In this chapter, the method of isotopologue profiling is illustrated with recent work on the metabolism of intracellular living Legionella pneumophila.

Mitochondrial Dynamics and Activity in Legionella-Infected Cells.

The study of Legionella pneumophila interactions with host mitochondria during infection has been historically limited by the techniques available to analyze and quantify mitochondrial dynamics and activity in living cells. Recently, new, powerful techniques such as high-content microscopy or mitochondrial respiration assays (Seahorse) have been developed to quantitatively analyze mitochondrial parameters. Here we present state-of-the-art methods adapted to analyze mitochondrial dynamics and activity during Legionella infection of living human primary macrophages.

Perturbation of Legionella Cell Infection by RNA Interference.

Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by RNA interference (RNAi) has proven to be a robust and economic technique to study L. pneumophila-host cell interactions. Predesigned and often validated double-stranded (ds) RNA oligonucleotides that silence specific genes are commercially available. RNAi results in a reduced level of distinct proteins, which allows studying the specific role of host cell components involved in L. pneumophila infection. Here, we describe how to assess RNAi-mediated protein depletion efficiency and cytotoxic effects in human A549 lung epithelial cells and murine RAW 264.7 macrophages. Moreover, we demonstrate how RNAi can be used to screen for novel host cell proteins involved in the formation of the Legionella-containing vacuole and intracellular replication of the pathogen.

Inflammasome Activation in Legionella-Infected Macrophages.

Legionella pneumophila is a gram-negative bacterium that infects many species of unicellular protozoa in freshwater environments. The human infection is accidental, and the bacteria may not have evolved strategies to bypass innate immune signaling in mammalian macrophages. Thus, L. pneumophila triggers many innate immune pathways including inflammasome activation. The inflammasomes are multimolecular platforms assembled in the host cell cytoplasm and lead to activation of inflammatory caspases. Inflammasome activation leads to secretion of inflammatory cytokines, such as IL-1ß and IL-18, and an inflammatory form of cell death called pyroptosis, which initiates with the induction of a pore in the macrophage membranes. In this chapter we provide detailed protocols to evaluate Legionella-induced inflammasome activation in macrophages, including real-time pore formation assay, western blotting to detect activation of inflammatory caspases (cleavage and pulldown), and the measurement of inflammatory cytokines.

The Galleria mellonella Infection Model for Investigating the Molecular Mechanisms of Legionella Virulence.

Legionella species evolved virulence factors to exploit protozoa as replicative niches in the environment. Cell culture infection models demonstrated that many of these factors also enable the bacteria to thrive in human macrophages; however, these models do not recapitulate the complex interactions between macrophages, lung epithelial, and additional immune cells, which are crucial to control bacterial infections. Thus, suitable infection models are required to understand which bacterial factors are important to trigger disease. Guinea pigs and, most frequently, mice have been successfully used as mammalian model hosts; however, ethical and economic considerations impede their use in high-throughput screening studies of Legionella isolates or small molecule inhibitors.Here, we describe the larvae of the lepidopteran Galleria mellonella as insect model of Legionella pathogenesis. Larvae can be obtained from commercial suppliers in large numbers, maintained without the need of specialized equipment, and infected by injection. Although lacking the complexity of a mammalian immune system, the larvae mount humoral and cellular immune responses to infection. L. pneumophila strain 130b and other prototype isolates withstand these responses and use the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) type IV secretion system (T4SS ) to inject effectors enabling survival and replication in hemocytes, insect phagocytes, ultimately leading to the death of the larvae. Differences in virulence between L. pneumophila isolates or gene deletion mutants can be analyzed using indicators of larval health and immune induction, such as pigmentation, mobility, histopathology, and survival. Bacterial replication can be measured by plating hemolymph or by immunofluorescence microscopy of isolated circulating hemocytes from infected larvae. Combined, these straightforward experimental readouts make G. mellonella larvae a versatile model host to rapidly assess the virulence of different Legionella isolates and investigate the role of specific virulence factors in overcoming innate host defense mechanisms.

A case report of Legionella and Mycoplasma pneumonia: Co-incidence or co-infection?

RATIONALE: Concurrent or sequential coinfections of Legionella pneumophila and Mycoplasma pneumoniae have been reported in the past though infrequently. Distinguishing a true co-infection from cross reactivity is often challenging as the diagnosis is mostly dependent on serological testing. PATIENT CONCERNS: A 77-year-old male presented with worsening dyspnea, cough with yellow sputum, diarrhea and fever of 2-days duration. Patient had history of chronic obstructive pulmonary disease (COPD) on home oxygen, bronchiectasis, rheumatoid arthritis (on methotrexate and leflunomide), treated pulmonary tuberculosis and 30-pack-year smoking. Chest X-ray showed bilateral interstitial changes with left lower lobe infiltrate. On day 5, his urine antigen for L pneumophila serogroup 1 was reported positive. The following day his serum M pneumoniae IgM antibody titers were reported elevated at 6647 U/mL. Patient was started on antibiotics and placed on non-invasive positive pressure ventilation. DIAGNOSIS: The patient was diagnosed with possible Legionella and Mycoplasma co-infection. OUTCOMES: Sputum Mycoplasma polymerase chain reaction (PCR) and serum cold agglutinins were obtained on day 6 and later reported negative. He was treated with azithromycin for 10 days with clinical improvement. LESSONS: Serological testing alone is an indirect measure with poor sensitivity and specificity and has its own limitations. Urine antigen detection confirms L pneumophila serogroup 1 infection in a patient with suggestive symptoms. However, diagnosis of M pneumonia should be based on combination of tests including serology and PCR to confirm true co-infection.