RESUMEN
Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.
Asunto(s)
Axonema , Cilios , Trastornos de la Motilidad Ciliar , Flagelos , Mecanotransducción Celular , Humanos , Masculino , Inteligencia Artificial , Dineínas Axonemales/química , Dineínas Axonemales/metabolismo , Dineínas Axonemales/ultraestructura , Axonema/química , Axonema/metabolismo , Axonema/ultraestructura , Cilios/química , Cilios/metabolismo , Cilios/ultraestructura , Microscopía por Crioelectrón , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestructura , Microtúbulos/metabolismo , Chlamydomonas reinhardtii , Trastornos de la Motilidad Ciliar/metabolismo , Trastornos de la Motilidad Ciliar/patología , Trastornos de la Motilidad Ciliar/fisiopatología , Movimiento , Conformación ProteicaRESUMEN
Here, we report that important regulators of cilia formation and ciliary compartment-directed protein transport function in secretion polarity. Mutations in cilia genes cep290 and bbs2, involved in human ciliopathies, affect apical secretion of Cochlin, a major otolith component and a determinant of calcium carbonate crystallization form. We show that Cochlin, defective in human auditory and vestibular disorder, DFNA9, is secreted from small specialized regions of vestibular system epithelia. Cells of these regions secrete Cochlin both apically into the ear lumen and basally into the basal lamina. Basally secreted Cochlin diffuses along the basal surface of vestibular epithelia, while apically secreted Cochlin is incorporated into the otolith. Mutations in a subset of ciliopathy genes lead to defects in Cochlin apical secretion, causing abnormal otolith crystallization and behavioral defects. This study reveals a class of ciliary proteins that are important for the polarity of secretion and delineate a secretory pathway that regulates biomineralization.
Asunto(s)
Ciliopatías/genética , Membrana Otolítica/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Síndrome de Bardet-Biedl/genética , Secuencia de Bases , Cilios/metabolismo , Cristalización , Epistasis Genética , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Mutación/genética , Fenotipo , Proteínas de Pez Cebra/genéticaRESUMEN
Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.
Asunto(s)
Transporte Biológico/genética , Flagelos/genética , Depuración Mucociliar/genética , Mutación/genética , Proteínas/genética , Adulto , Alelos , Axonema/genética , Línea Celular , Chlamydomonas/genética , Cilios/genética , Dineínas/genética , Células Epiteliales/patología , Femenino , Células HEK293 , Humanos , Lactante , Masculino , Fenotipo , Trypanosoma brucei brucei/genéticaRESUMEN
Motile cilia move body fluids and gametes and the beating of cilia lining the airway epithelial surfaces ensures that they are kept clear and protected from inhaled pathogens and consequent respiratory infections. Dynein motor proteins provide mechanical force for cilia beating. Dynein mutations are a common cause of primary ciliary dyskinesia (PCD), an inherited condition characterized by deficient mucociliary clearance and chronic respiratory disease coupled with laterality disturbances and subfertility. Using next-generation sequencing, we detected mutations in the ciliary outer dynein arm (ODA) heavy chain gene DNAH9 in individuals from PCD clinics with situs inversus and in one case male infertility. DNAH9 and its partner heavy chain DNAH5 localize to type 2 ODAs of the distal cilium and in DNAH9-mutated nasal respiratory epithelial cilia we found a loss of DNAH9/DNAH5-containing type 2 ODAs that was restricted to the distal cilia region. This confers a reduced beating frequency with a subtle beating pattern defect affecting the motility of the distal cilia portion. 3D electron tomography ultrastructural studies confirmed regional loss of ODAs from the distal cilium, manifesting as either loss of whole ODA or partial loss of ODA volume. Paramecium DNAH9 knockdown confirms an evolutionarily conserved function for DNAH9 in cilia motility and ODA stability. We find that DNAH9 is widely expressed in the airways, despite DNAH9 mutations appearing to confer symptoms restricted to the upper respiratory tract. In summary, DNAH9 mutations reduce cilia function but some respiratory mucociliary clearance potential may be retained, widening the PCD disease spectrum.
Asunto(s)
Dineínas Axonemales/genética , Cilios/genética , Dineínas/genética , Mutación/genética , Situs Inversus/genética , Adolescente , Secuencia de Aminoácidos , Niño , Preescolar , Trastornos de la Motilidad Ciliar/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Sistema Respiratorio/patología , Alineación de SecuenciaRESUMEN
BACKGROUND: Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies. METHODS: We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. RESULTS: Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. CONCLUSION: Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.
Asunto(s)
Trastornos de la Motilidad Ciliar , Síndrome de Kartagener , Cilios , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/tratamiento farmacológico , Trastornos de la Motilidad Ciliar/genética , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/tratamiento farmacológico , Síndrome de Kartagener/genética , Depuración MucociliarRESUMEN
BACKGROUND: Primary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests. METHODS: The diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries. RESULTS: Most (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results. CONCLUSIONS: This study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening.
Asunto(s)
Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Alelos , Pueblo Asiatico/genética , Cilios/patología , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/patología , Estudios de Cohortes , Etnicidad/genética , Femenino , Homocigoto , Humanos , Masculino , Mutación/genética , FenotipoRESUMEN
A short-cut review of the literature was carried out to examine the diagnostic test characteristics and potential patient benefits through the use of the Ottawa Subarachnoid Haemorrhage Clinical Decision Rule. Nine papers were identified as suitable for inclusion using the reported search strategy. The author, date and country of publication, patient group studied, study type, relevant outcomes, results and study weaknesses of the best papers are tabulated. It is concluded that the Ottawa Clinical Decision Rule has a high sensitivity for the diagnosis of subarachnoid haemorrhage; however, there is limited robust evidence of international generalisability and no evidence of improved patient outcomes following implementation. Further prospective research is required in populations with variable prevalence to evaluate the safety and effectiveness of this intervention, compared with routine evaluation strategies.
Asunto(s)
Reglas de Decisión Clínica , Hemorragia Subaracnoidea/diagnóstico , Diagnóstico Diferencial , Medicina de Emergencia Basada en la Evidencia , Humanos , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To quantify psychological distress experienced by emergency, anaesthetic and intensive care doctors during the acceleration phase of COVID-19 in the UK and Ireland. METHODS: Initial cross-sectional electronic survey distributed during acceleration phase of the first pandemic wave of COVID-19 in the UK and Ireland (UK: 18 March 2020-26 March 2020 and Ireland: 25 March 2020-2 April 2020). Surveys were distributed via established specialty research networks, within a three-part longitudinal study. Participants were doctors working in emergency, anaesthetic and intensive medicine during the first pandemic wave of COVID-19 in acute hospitals across the UK and Ireland. Primary outcome measures were the General Health Questionnaire-12 (GHQ-12). Additional questions examined personal and professional characteristics, experiences of COVID-19 to date, risk to self and others and self-reported perceptions of health and well-being. RESULTS: 5440 responses were obtained, 54.3% (n=2955) from emergency medicine and 36.9% (n=2005) from anaesthetics. All levels of doctor seniority were represented. For the primary outcome of GHQ-12 score, 44.2% (n=2405) of respondents scored >3, meeting the criteria for psychological distress. 57.3% (n=3045) had never previously provided clinical care during an infectious disease outbreak but over half of respondents felt somewhat prepared (48.6%, n=2653) or very prepared (7.6%, n=416) to provide clinical care to patients with COVID-19. However, 81.1% (n=4414) either agreed (31.1%, n=2709) or strongly agreed (31.1%, n=1705) that their personal health was at risk due to their clinical role. CONCLUSIONS: Findings indicate that during the acceleration phase of the COVID-19 pandemic, almost half of frontline doctors working in acute care reported psychological distress as measured by the GHQ-12. Findings from this study should inform strategies to optimise preparedness and explore modifiable factors associated with increased psychological distress in the short and long term. TRIAL REGISTRATION NUMBER: ISRCTN10666798.
Asunto(s)
COVID-19/epidemiología , Medicina de Emergencia/estadística & datos numéricos , Estrés Laboral/epidemiología , Médicos/estadística & datos numéricos , Adulto , Anciano , Anestesia/estadística & datos numéricos , COVID-19/psicología , Cuidados Críticos/estadística & datos numéricos , Estudios Transversales , Femenino , Humanos , Irlanda/epidemiología , Masculino , Persona de Mediana Edad , Estrés Laboral/etiología , Médicos/psicología , Distrés Psicológico , Encuestas y Cuestionarios , Reino Unido/epidemiología , Adulto JovenRESUMEN
Impaired mucociliary clearance may increase COPD exacerbation risk. We aimed to compare bronchial ciliary function and epithelial ultrastructure of COPD patients to healthy controls and explore its relationship to exacerbator phenotypes (frequent [FE] and infrequent [IFE] exacerbator). In this cross-sectional study, 16 COPD patients and 12 controls underwent bronchial brushings. Ciliary beat frequency (CBF) and dyskinesia index (DI; % of dyskinetic cilia) were assessed using digital high-speed video microscopy, and epithelial ultrastructure using transmission electron microscopy (TEM). Bronchial epithelium in COPD showed lower CBF and higher DI, compared to controls (median [IQR] CBF: 6.8 (6.1-7.2) Hz vs 8.5 (7.7-8.9) Hz, p<0.001 and DI: 73.8 (60.7-89.8) % vs 14.5 (11.2-16.9) %, p<0.001, respectively). This was true for FE and IFE phenotypes of COPD, which were similar in terms of bronchial CBF or DI. Subgroup analyses demonstrated lower CBF and higher DI in FE and IFE COPD phenotypes compared to controls, irrespective of smoking status. TEM showed more loss of cilia, extrusion of cells, cytoplasmic blebs and dead cells in COPD patients versus controls. Profound dysfunction of bronchial cilia is a feature of COPD irrespective of exacerbation phenotype and smoking status, which is likely to contribute to poor mucus clearance in COPD.Supplemental data for this article is available online at https://doi.org/10.1080/15412555.2021.1963695 .
Asunto(s)
Cilios , Enfermedad Pulmonar Obstructiva Crónica , Bronquios , Cilios/ultraestructura , Estudios Transversales , Humanos , Mucosa RespiratoriaRESUMEN
Primary ciliary dyskinesia (PCD) is a heterogeneous genetic condition. European and North American diagnostic guidelines recommend transmission electron microscopy (TEM) as one of a combination of tests to confirm a diagnosis. However, there is no definition of what constitutes a defect or consensus on reporting terminology. The aim of this project was to provide an internationally agreed ultrastructural classification for PCD diagnosis by TEM.A consensus guideline was developed by PCD electron microscopy experts representing 18 centres in 14 countries. An initial meeting and discussion were followed by a Delphi consensus process. The agreed guideline was then tested, modified and retested through exchange of samples and electron micrographs between the 18 diagnostic centres.The final guideline a) provides agreed terminology and a definition of Class 1 defects which are diagnostic for PCD; b) identifies Class 2 defects which can indicate a diagnosis of PCD in combination with other supporting evidence; c) describes features which should be included in a ciliary ultrastructure report to assist multidisciplinary diagnosis of PCD; and d) defines adequacy of a diagnostic sample.This tested and externally validated statement provides a clear guideline for the diagnosis of PCD by TEM which can be used to standardise diagnosis internationally.
Asunto(s)
Trastornos de la Motilidad Ciliar , Síndrome de Kartagener , Cilios , Ingestión de Alimentos , Humanos , Síndrome de Kartagener/diagnóstico , Microscopía Electrónica , Microscopía Electrónica de TransmisiónRESUMEN
Primary ciliary dyskinesia (PCD) is associated with abnormal organ positioning (situs) and congenital heart disease (CHD). This study investigated genotype-phenotype associations in PCD to facilitate risk predictions for cardiac and laterality defects. This retrospective cohort study of 389 UK patients with PCD found 51% had abnormal situs and 25% had CHD and/or laterality defects other than situs inversus totalis. Patients with biallelic mutations in a subset of nine PCD genes had normal situs. Patients with consanguineous parents had higher odds of situs abnormalities than patients with non-consanguineous parents. Patients with abnormal situs had higher odds of CHD and/or laterality defects.
Asunto(s)
Anomalías Múltiples/epidemiología , Trastornos de la Motilidad Ciliar/epidemiología , Cardiopatías Congénitas/epidemiología , Situs Inversus/epidemiología , Anomalías Múltiples/genética , Trastornos de la Motilidad Ciliar/genética , Consanguinidad , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Cardiopatías Congénitas/genética , Humanos , Masculino , Mutación , Fenotipo , Prevalencia , Estudios Retrospectivos , Factores de Riesgo , Situs Inversus/genética , Reino Unido/epidemiologíaRESUMEN
RATIONALE: Primary ciliary dyskinesia is a genetically heterogeneous inherited condition characterised by progressive lung disease arising from abnormal cilia function. Approximately half of patients have situs inversus. The estimated prevalence of primary ciliary dyskinesia in the UK South Asian population is 1:2265. Early, accurate diagnosis is key to implementing appropriate management but clinical diagnostic tests can be equivocal. OBJECTIVES: To determine the importance of genetic screening for primary ciliary dyskinesia in a UK South Asian population with a typical clinical phenotype, where standard testing is inconclusive. METHODS: Next-generation sequencing was used to screen 86 South Asian patients who had a clinical history consistent with primary ciliary dyskinesia. The effect of a CCDC103 p.His154Pro missense variant compared with other dynein arm-associated gene mutations on diagnostic/phenotypic variability was tested. CCDC103 p.His154Pro variant pathogenicity was assessed by oligomerisation assay. RESULTS: Sixteen of 86 (19%) patients carried a homozygous CCDC103 p.His154Pro mutation which was found to disrupt protein oligomerisation. Variable diagnostic test results were obtained including normal nasal nitric oxide levels, normal ciliary beat pattern and frequency and a spectrum of partial and normal dynein arm retention. Fifteen (94%) patients or their sibling(s) had situs inversus suggesting CCDC103 p.His154Pro patients without situs inversus are missed. CONCLUSIONS: The CCDC103 p.His154Pro mutation is more prevalent than previously thought in the South Asian community and causes primary ciliary dyskinesia that can be difficult to diagnose using pathology-based clinical tests. Genetic testing is critical when there is a strong clinical phenotype with inconclusive standard diagnostic tests.
Asunto(s)
Pueblo Asiatico/genética , Síndrome de Kartagener/etnología , Síndrome de Kartagener/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación/genética , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Masculino , Pakistán/etnología , Reino Unido , Adulto JovenRESUMEN
BACKGROUND: Primary ciliary dyskinesia can result from a number of different ciliary defects that adversely affect ciliary function resulting markedly reduced or absent mucociliary clearance. Improvement in diagnostic testing is an area of current research. During diagnostic evaluation of PCD we observed ciliated conical protrusions from part of the apical surface of ciliated cells in those diagnosed with PCD. The aim of this study was to investigate if this abnormality was specific to PCD. METHODS: Epithelial edges from 67 consecutively diagnosed PCD patients, 67 patients consecutively referred for PCD diagnostic testing in whom PCD was excluded, 22 with asthma and 18 with Cystic Fibrosis (CF) were studied retrospectively in a blinded manner using light microscopy. RESULTS: Forty six out of 67 patients with PCD had ciliated conical epithelial protrusions, whereas none were seen in patients where PCD was excluded, or in patients with asthma or CF. The sensitivity, specificity, positive predictive value and negative predictive value for the presence of the ciliated conical protrusions to predict a diagnosis of PCD were 76.5, 100, 100 and 77% respectively. CONCLUSIONS: Characteristic ciliated conical protrusions from ciliated epithelial cells maybe a useful pointer to the diagnosis of PCD. However, their absence does not exclude the diagnosis of PCD.
Asunto(s)
Cilios/patología , Cilios/fisiología , Síndrome de Kartagener/patología , Depuración Mucociliar/fisiología , Mucosa Respiratoria/patología , Mucosa Respiratoria/fisiología , Células Cultivadas , HumanosAsunto(s)
Charadriiformes , Medicina de Emergencia , Humanos , Animales , Irlanda , Cuidadores , Técnica Delphi , Investigación , Reino UnidoRESUMEN
Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse Bmi-1 + hTERT, but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of human BMI-1 alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. Cystic fibrosis (CF) and non-CF bronchial epithelial cells were transduced by lentivirus with BMI-1 and then their morphology, replication kinetics, and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function, and transepithelial electrophysiology were measured. Finally, shRNA knockdown of DNAH5 in BMI-1 cells was used to model primary ciliary dyskinesia (PCD). BMI-1-transduced basal cells showed normal cell morphology, karyotype, and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics of BMI-1-transduced cells were similar to those of untransduced cells. shRNA knockdown of DNAH5 in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination.
Asunto(s)
Bronquios/citología , Diferenciación Celular , Cilios/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Moco/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Animales , Dineínas Axonemales/metabolismo , Proliferación Celular , Forma de la Célula , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Dineínas/metabolismo , Impedancia Eléctrica , Fenómenos Electrofisiológicos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patología , Síndrome de Kartagener/fisiopatología , Cariotipificación , Ratones , Microtúbulos/metabolismo , Modelos Biológicos , Fenotipo , Transducción GenéticaRESUMEN
The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no "gold standard" reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia.
Asunto(s)
Cilios/ultraestructura , Síndrome de Kartagener/diagnóstico , Cilios/patología , Técnica Delphi , Diagnóstico Diferencial , Europa (Continente) , Técnica del Anticuerpo Fluorescente , Pruebas Genéticas , Humanos , Síndrome de Kartagener/genética , Microscopía Electrónica de Transmisión , Microscopía por Video , Óxido Nítrico/análisis , Literatura de Revisión como Asunto , Sociedades MédicasRESUMEN
RATIONALE: Respiratory syncytial virus (RSV) is a highly contagious pathogen with a huge global health impact. It is a major cause of hospital-acquired infection; a large number of those exposed develop infection. Those infected in hospital are at increased risk of a severe clinical course. Prevention of nosocomial spread currently focuses on spread by hand and large droplets. There is little research evidence to determine if aerosol spread of infectious RSV is possible. OBJECTIVES: To determine if the air surrounding infants with RSV-positive bronchiolitis contains RSV in aerosolized particles that remain capable of causing infection. METHODS: The amount of RSV contained in aerosolized particles produced by infants with bronchiolitis due to RSV was measured using viable impactor sampling. The ability of RSV contained in these particles to infect healthy and chronic obstructive pulmonary disease (COPD) human ciliated respiratory epithelium was determined. RESULTS: We showed for the first time that infants with RSV-positive bronchiolitis nursed in a ward setting or ventilated in intensive care produced large numbers of aerosol particles containing RSV that remained infectious and were capable of infecting healthy and COPD human ciliated epithelium. A significant amount of RSV was found in particles with aerodynamic diameters less than 5 µm. CONCLUSIONS: Many of the aerosolized particles that contained RSV in the air surrounding infants with bronchiolitis were sufficiently small to remain airborne for a significant length of time and small enough to be inhaled and deposited throughout the respiratory tract. It is likely that this leads to spread of infection to others, with dissemination of infection throughout the respiratory tract.