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BackgroundPrimary ciliary dyskinesia (PCD) is a heterogeneous inherited disorder caused by mutations in approximately 50 cilia-related genes. PCD genotype–phenotype relationships have mostly arisen from small case series because existing statistical approaches to investigating relationships have been unsuitable for rare diseases.MethodsWe applied a topological data analysis (TDA) approach to investigate genotype–phenotype relationships in PCD. Data from separate training and validation cohorts included 396 genetically defined individuals carrying pathogenic variants in PCD genes. To develop the TDA models, 12 clinical and diagnostic variables were included. TDA-driven hypotheses were subsequently tested using traditional statistics.ResultsDisease severity at diagnosis, measured by forced expiratory volume in 1 s (FEV1) z-score, was significantly worse in individuals with CCDC39 mutations (compared to other gene mutations) and better in those with DNAH11 mutations; the latter also reported less neonatal respiratory distress. Patients without neonatal respiratory distress had better preserved FEV1 at diagnosis. Individuals with DNAH5 mutations were phenotypically diverse. Cilia ultrastructure and beat pattern defects correlated closely to specific causative gene groups, confirming these tests can be used to support a genetic diagnosis.ConclusionsThis large scale, multi-national study presents PCD as a syndrome with overlapping symptoms and variations in phenotype according to genotype. TDA modelling confirmed genotype–phenotype relationships reported by smaller studies (e.g. FEV1 worse with CCDC39 mutation) and identified new relationships, including FEV1 preservation with DNAH11 mutations and diversity of severity with DNAH5 mutations.

Air&ndash, liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9%) were ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen, 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.

ObjectiveIn England, the National Health Service commissioned a National Management Service for children with primary ciliary dyskinesia (PCD). The aims of this study were to describe the health of children seen in this Service and compare lung function to children with cystic fibrosis (CF).DesignMulti-centre service evaluation of the English National Management PCD Service.SettingFour nationally commissioned PCD centres in England.Patients333 children with PCD reviewed in the Service in 2015; lung function data were also compared with 2970 children with CF.ResultsMedian age at diagnosis for PCD was 2.6 years, significantly lower in children with situs inversus (1.0 vs 6.0 years, p1) was 76.8% in PCD (n=240) and 85.0% in CF, and FEV1 was lower in children with PCD up to the age of 15 years. Approximately half of children had some hearing impairment, with 26% requiring hearing aids. Children with a lower body mass index (BMI) had lower FEV1 (pHaemophilus influenzae.ConclusionsWe provide evidence that children with PCD in England have worse lung function than those with CF. Nutritional status should be considered in PCD management, as those with a lower BMI have significantly lower FEV1. Hearing impairment is common but seems to improve with age. Well-designed and powered randomised controlled trials on management of PCD are needed to inform best clinical practice.

We welcome the correspondence from Lavie and Amirav (1), highlighting the difficulties diagnosing primary ciliary dyskinesia (PCD) and the role of high-speed video analysis (HSVA). As members of the European Respiratory Society (ERS) PCD Diagnostic Task Force (2) and/or large PCD Centres, we agree that HSVA has an important role that is not recognized by the American Thoracic Society (ATS) PCD Diagnostic Guideline (3). This risks a large proportion of false-negative “missed” diagnoses and a sizable number of false-positive cases; we make additional important observations.

Primary ciliary dyskinesia (PCD) is a rare heterogenous condition that causes progressive suppurative lung disease, chronic rhinosinusitis, chronic otitis media, infertility and abnormal situs. ‘Better Experimental Approaches to Treat Primary Ciliary Dyskinesia’ (BEAT-PCD) is a network of scientists and clinicians coordinating research from basic science through to clinical care with the intention of developing treatments and diagnostics that lead to improved long-term outcomes for patients. BEAT-PCD activities are supported by EU funded COST Action (BM1407). The second BEAT-PCD conference, and third PCD training school were held jointly in April 2017 in Valencia, Spain. Presentations and workshops focussed on advancing the knowledge and skills relating to PCD in: basic science, epidemiology, diagnostic testing, clinical management and clinical trials. The multidisciplinary conference provided an interactive platform for exchanging ideas through a program of lectures, poster presentations, breakout sessions and workshops. Three working groups met to plan consensus statements. Progress with BEAT-PCD projects was shared and new collaborations were fostered. In this report, we summarize the meeting, highlighting developments made during the meeting. Electronic supplementary material The online version of this article (10.1186/s12919-018-0098-9) contains supplementary material, which is available to authorized users.

International audience; 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.

Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive condition affecting around 1:15,000. In people with PCD, microscopic motile cilia do not move normally resulting in impaired clearance of mucus and debris leading to repeated sinopulmonary infection. If diagnosis is delayed, permanent bronchiectasis and deterioration of lung function occurs. Other complications associated with PCD include congenital heart disease, hearing impairment and infertility. A small number of longitudinal studies suggest that lung function deteriorates before diagnosis of PCD but may stabilise following diagnosis with subsequent specialist management. Early diagnosis is therefore essential, but for a number of reasons referral for diagnostic testing is often delayed until older childhood or even adulthood. Functional diagnostic tests for PCD are expensive, time consuming and require specialist equipment and scientists. In the last few years, there have been considerable developments to identify genes associated with PCD, currently enabling 65% of patients to be identified by bi-allelic mutations. The rapid identification of new genes continues. This review will consider the evidence that early diagnosis of PCD is beneficial. It will review the recent advances in identification of PCD-associated genes and will discuss the role of genetic testing in PCD. It will then consider whether screening for PCD antenatally or in the new born is likely to become a feasible and acceptable for this rare disease.

PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability ( P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed ( P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.

Diagnosis of primary ciliary dyskinesia (PCD) lacks a “gold standard” test and is therefore based on combinations of tests including nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA), genotyping and transmission electron microscopy (TEM). There are few published data on the accuracy of this approach. Using prospectively collected data from 654 consecutive patients referred for PCD diagnostics we calculated sensitivity and specificity for individual and combination testing strategies. Not all patients underwent all tests. HSVMA had excellent sensitivity and specificity (100% and 93%, respectively). TEM was 100% specific, but 21% of PCD patients had normal ultrastructure. nNO (30 nL·min−1 cut-off) had good sensitivity and specificity (91% and 96%, respectively). Simultaneous testing using HSVMA and TEM was 100% sensitive and 92% specific. In conclusion, combination testing was found to be a highly accurate approach for diagnosing PCD. HSVMA alone has excellent accuracy, but requires significant expertise, and repeated sampling or cell culture is often needed. TEM alone is specific but misses 21% of cases. nNO (≤30 nL·min−1) contributes well to the diagnostic process. In isolation nNO screening at this cut-off would miss ∼10% of cases, but in combination with HSVMA could reduce unnecessary further testing. Standardisation of testing between centres is a future priority., Combination testing in PCD diagnosis remains the most accurate approach, but standardisation is needed http://ow.ly/TLEDu

Symptoms of primary ciliary dyskinesia (PCD) are nonspecific and guidance on whom to refer for testing is limited. Diagnostic tests for PCD are highly specialised, requiring expensive equipment and experienced PCD scientists. This study aims to develop a practical clinical diagnostic tool to identify patients requiring testing. Patients consecutively referred for testing were studied. Information readily obtained from patient history was correlated with diagnostic outcome. Using logistic regression, the predictive performance of the best model was tested by receiver operating characteristic curve analyses. The model was simplified into a practical tool (PICADAR) and externally validated in a second diagnostic centre. Of 641 referrals with a definitive diagnostic outcome, 75 (12%) were positive. PICADAR applies to patients with persistent wet cough and has seven predictive parameters: full-term gestation, neonatal chest symptoms, neonatal intensive care admittance, chronic rhinitis, ear symptoms, situs inversus and congenital cardiac defect. Sensitivity and specificity of the tool were 0.90 and 0.75 for a cut-off score of 5 points. Area under the curve for the internally and externally validated tool was 0.91 and 0.87, respectively. PICADAR represents a simple diagnostic clinical prediction rule with good accuracy and validity, ready for testing in respiratory centres referring to PCD centres., PICADAR is a simple diagnostic prediction tool for PCD with good accuracy and validity that is now ready for testing http://ow.ly/X6y9s

Introduction: Pandemic influenza A H1N1 infections occurred worldwide from 2009. Children were particularly vulnerable. Novel vaccines were used during the pandemic. Objective: To assess the persistence of antibody to H1N1 influenza 1 year after children aged 6 months to 12 years had been immunised with two doses of either a non-adjuvanted whole-virion H1N1 influenza vaccine or an AS03 B-adjuvanted split-virion H1N1 influenza vaccine; and also to assess the immunogenicity and reactogenicity in this population of a single dose of 2010-11 trivalent seasonal influenza vaccine. Design: Multicentre, open-label, follow-on from randomised, head-to-head trial. Setting: Five UK sites (Southampton, Oxford, Bristol, London and Exeter). Participants: Children who completed last year's head-to-head randomised study were invited to participate. Children who had subsequently received a further dose of H1N1 vaccine, or who had already received a dose of 2010-11 trivalent seasonal influenza vaccine, were excluded. Interventions: In the previous study, children were randomised (in a 1: 1 ratio) to receive two doses, 21 days apart, of either a non-adjuvanted whole-virion H1N1 influenza vaccine or an AS03 B-adjuvanted split-virion H1N1 influenza vaccine. In this follow-on study, a blood sample was taken to assess the persistence of antibody 1 year later, followed by administration of one 0.5 ml-dose of trivalent seasonal influenza vaccine. A second blood sample was taken 3 weeks later. Main outcome measures: Comparison between vaccines of the percentage of participants with a microneutralisation (MN) titre ≥ 1: 40 and a haemagglutination titre ≥ 1: 32, 1 year after vaccination. Immunogenicity of the trivalent seasonal influenza vaccine was assessed 3 weeks after vaccination by both the MN and the haemagglutination inhibition (HI) titres. Reactogenicity data were recorded for 7 days after vaccination. Results: A total of 323 children were enrolled and 318 were included in the analysis of the persistence of antibody. One year after receipt of whole-virion vaccine, the MN titre was ≥ 1: 40 in 32.4% of those vaccinated when < 3 years old and in 65.9% of those vaccinated when ≥ 3 years old; the HI titre was ≥ 1: 32 in 63.2% and 79.1% of children in the respective age groups. One year after receipt of the adjuvanted vaccine, the MN titre was ≥ 1: 40 in 100% of those vaccinated when < 3 years old and in 96.9% of those vaccinated when ≥ 3 years old; the HI titre was ≥ 1: 32 in 98.4% and 96.9% of children in the respective age groups. Three hundred and two children were given trivalent seasonal influenza vaccination. Three weeks later, sera were obtained from 282 children; 100% had an MN titre ≥ 1: 40 and HI titre ≥ 1: 32. Trivalent seasonal influenza vaccine was well tolerated, although in children < 5 years old, fever ≥ 38 °C was reported in 13.6% of those who had previously received whole-virion vaccine, and in 18.3% of those who had received adjuvanted vaccine. Conclusions: Nearly all children who received two doses of AS03 B-adjuvanted split-virion pandemic H1N1 influenza vaccine had titres of antibody deemed protective (HI titre ≥ 1: 32, MN titre ≥ 1: 40) 1 year later. Children who received two doses of whole-virion vaccine had lower titres, although many were above the putative protective thresholds. One year after either pandemic vaccine, the 2010-11 trivalent seasonal influenza vaccine produced a marked serological response to the H1N1 component of the vaccine and was well tolerated. We propose to investigate whether or not previous receipt of monovalent influenza vaccines affected serological response to the H3N2 and B components of the 2010-11 seasonal influenza vaccine, using stored sera. Trial registration: ClinicalTrials.gov NCT01239537. Funding: The National Institute for Health Research Health Technology Assessment programme. © Queen's Printer and Controller of HMSO 2011.

In 2009, 943 children aged 6 months to 10 years were randomised to receive two doses of an oil-in water AS03B-adjuvanted split virion or a non-adjuvanted whole virion H1N1 (2009) vaccine. The large numbers allowed investigation of possible predictors of immune response and reactogenicity. We used regression analysis to examine the effect of variables including past receipt of seasonal vaccine, antipyretics post-vaccination, interval between doses and pre-existing antibodies to H1N1 (2009) on immunogenicity. We also examined the relationship between immunogenicity and reactogenicity and whether prior infection or underlying conditions affected reactogenicity. For both vaccines, haemagglutination-inhibition titres were 60% higher in children with fever ≥38. °C after vaccination and 29% lower in those previously given seasonal vaccine. Early use of antipyretics did not affect immunogenicity. Post-vaccination titres were higher with longer intervals between doses and in those with evidence of prior infection, but reactogenicity in the latter was unaffected. In the adjuvanted vaccine group, reactions were more common in children with atopy. Both vaccines were safe and immunogenic in those with prior infection. Reduction in the interval between doses for earlier protection would be at the cost of reduced immunogenicity. The effect of seasonal vaccine on immunogenicity merits further investigation. © 2011 Elsevier Ltd.

Background: Children are a priority for vaccination in an influenza pandemic, but safety and immunogenicity data for new-generation adjuvanted and whole-virion vaccines are limited. Objectives: Immunogenicity •How does the percentage of children aged 6 months to 12 years of age with a fourfold rise in microneutralisation titres between the prevaccination sample and the sample taken 3 weeks after completion of a two-dose course of the non-adjuvanted, whole-virion vaccine and the AS03B-adjuvanted split-virion vaccine compare? •How does the percentage of children aged 6 months to 12 years of age with haemagglutination inhibition titres of ? 1 : 32 3 weeks after completion of a two-dose course of the non-adjuvanted, whole-virion vaccine and the AS03B-adjuvanted split-virion vaccine compare? •How does the percentage of children aged 6 months to 12 years of age with a fourfold rise in haemagglutination inhibition titres between the prevaccination sample and the sample taken 3 weeks after completion of a two-dose course of the non-adjuvanted, whole-virion vaccine and the AS03B-adjuvanted split-virion vaccine compare? •What is the geometric mean fold rise in haemagglutination inhibition titres from baseline to 3 weeks after two doses of the non-adjuvanted, whole-virion vaccine and the AS03B-adjuvanted split-virion vaccine? •What is the geometric mean haemagglutination inhibition titre 3 weeks after two doses of the non-adjuvanted, whole-virion vaccine and the AS03B-adjuvanted split-virion vaccine? Reactogenicity •How does the percentage of children aged 6 months to 12 years of age experiencing fever and local reactions within the 7 days following each dose of the non-adjuvanted, whole-virion and the AS03B-adjuvanted split-virion vaccines compare? •What percentage of children aged 6 months to 12 years of age experience non-febrile systemic reactions within the 7 days following each dose of the non-adjuvanted, whole-virion and the AS03B-adjuvanted split-virion vaccine? Methods: The safety, reactogenicity and immunogenicity of a tocopherol/oil-in-water emulsion-adjuvanted (AS03B) egg culture-derived split-virion H1N1 vaccine and a non-adjuvanted cell culture-derived whole-virion vaccine, given as a two-dose schedule, 21 days apart, were compared in a randomised, open-label trial of children aged 6 months to 12 years of age. Local reactions and systemic symptoms were collected for 1 week post immunisation, and serum was collected at baseline and after the second dose. Results: Among 937 children receiving vaccine, per-protocol seroconversion rates were higher after the AS03B-adjuvanted vaccine than after the whole-virion vaccine (98.2% vs 80.1% in children 38.0°C in those under 5 years of age (8.9% vs 22.4%). Conclusion: In this first direct comparison of an AS03B-adjuvanted split-virion vaccine versus whole-virion non-adjuvanted H1N1 vaccine, the adjuvanted vaccine – while reactogenic – was more immunogenic, especially in younger children, indicating the potential for improved immunogenicity of influenza vaccines in this age group.

OBJECTIVES: To compare the safety, reactogenicity, and immunogenicity of an adjuvanted split virion H1N1 vaccine and a non-adjuvanted whole virion vaccine used in the pandemic immunisation programme in the United Kingdom. DESIGN: Open label, randomised, parallel group, phase II study. SETTING: Five UK centres (Oxford, Southampton, Bristol, Exeter, and London). PARTICIPANTS: Children aged 6 months to less than 13 years for whom a parent or guardian had provided written informed consent and who were able to comply with study procedures were eligible. Those with laboratory confirmed pandemic H1N1 influenza or clinically diagnosed disease meriting antiviral treatment, allergy to egg or any other vaccine components, or coagulation defects, or who were severely immunocompromised or had recently received blood products were excluded. Children were grouped by age: 6 months-or=1:40 from before vaccination to three weeks after the second dose). RESULTS: Seroconversion rates were higher after the adjuvanted split virion vaccine than after the whole virion vaccine, most notably in the youngest children (163 of 166 participants with paired serum samples (98.2%, 95% confidence interval 94.8% to 99.6%) v 157 of 196 (80.1%, 73.8% to 85.5%), Por=38 masculineC in those aged under 5 (24 (8.9%, 5.8% to 12.9%) v 57 (22.4%, 17.5% to 28.1%), P

Summary Background Patients with primary ciliary dyskinesia (PCD) have abnormal ciliary function and low nitric oxide levels. Nitric oxide (NO) biosynthesis is dependent on nitric oxide synthases (NOS). Cilia line the bronchial but not the alveolar epithelium. It has been hypothesised that NOS function relies on normal ciliary function and that in PCD bronchial but not alveolar NO might therefore be reduced. The aim of this study was to assess bronchial and alveolar NO levels primarily comparing healthy children to PCD and secondarily to cystic fibrosis (CF) and asthmatic children. Methods Multiple flow-rate fractional exhaled and nasal NO measurements were performed using a NIOX ® Flex NO analyser (Aerocrine, Sweden) in children with PCD ( n = 14), asthma ( n = 18), CF ( n = 12) and healthy controls ( n = 18). Alveolar and bronchial NO levels were derived using a model of pulmonary NO exchange-dynamics. Results Both the bronchial and alveolar NO were significantly lower in PCD than healthy controls (mean (SD) 264 (209) picolitres/second (pl/s) vs. 720 (514) pl/s, p = 0.024 and 1.7 (0.8) parts per billion (ppb) vs. 3.5 (1.3) ppb, p = 0.001 respectively.) In asthmatics bronchial NO was found to be significantly higher than in healthy controls and in children with CF alveolar NO was significantly lower (2100 (1935) pl/s, p = 0.045 and 2.5 (1.2) ppb, p = 0.034 respectively.) Conclusion Our findings do not support the hypothesis that NOS and ciliary function are coupled instead suggesting a more generalised mechanism for the low levels of NO seen in PCD. Our findings in CF and asthma corroborate evidence that these are diseases of the lung peripheries and bronchi respectively.

Background Patients with primary ciliary dyskinesia (PCD) have abnormal ciliary function and low nasal nitric oxide (nNO) and fractional exhaled NO (FeNO). Nitric oxide (NO) biosynthesis is dependent on nitric oxide synthases (NOS). Cilia line the bronchial but not the alveolar epithelium. It has been hypothesised that NOS function relies on normal ciliary function and that in PCD bronchial (JNO) but not alveolar (CalvNO) NO might therefore be reduced. The aim of this study was to compare JNO and CalvNO in children with PCD, cystic fibrosis (CF), asthma and healthy subjects.