A Comprehensive Approach for the Diagnosis of Primary Ciliary Dyskinesia—Experiences from the First 100 Patients of the PCD-UNIBE Diagnostic Center

Primary ciliary dyskinesia (PCD) is a rare genetic disease characterized by dyskinetic cilia. Respiratory symptoms usually start at birth. The lack of diagnostic gold standard tests is challenging, as PCD diagnostics requires different methods with high expertise. We founded PCD-UNIBE as the first comprehensive PCD diagnostic center in Switzerland. Our diagnostic approach includes nasal brushing and cell culture with analysis of ciliary motility via high-speed-videomicroscopy (HSVM) and immunofluorescence labeling (IF) of structural proteins. Selected patients undergo electron microscopy (TEM) of ciliary ultrastructure and genetics. We report here on the first 100 patients assessed by PCD-UNIBE. All patients received HSVM fresh, IF, and cell culture (success rate of 90%). We repeated the HSVM with cell cultures and conducted TEM in 30 patients and genetics in 31 patients. Results from cell cultures were much clearer compared to fresh samples. For 80 patients, we found no evidence of PCD, 17 were diagnosed with PCD, two remained inconclusive, and one case is ongoing. HSVM was diagnostic in 12, IF in 14, TEM in five and genetics in 11 cases. None of the methods was able to diagnose all 17 PCD cases, highlighting that a comprehensive approach is essential for an accurate diagnosis of PCD.

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Faculty Opinions recommendation of Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.721627008.793504018 ◽

2015 ◽

Author(s):

Nicholas Katsanis

Keyword(s):

Primary Ciliary Dyskinesia ◽

Ciliary Dyskinesia ◽

Ciliary Ultrastructure

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Diagnosis of primary ciliary dyskinesia: summary of the ERS Task Force report

Breathe ◽

10.1183/20734735.008517 ◽

2017 ◽

Vol 13 (3) ◽

pp. 166-178 ◽

Cited By ~ 23

Author(s):

Claudia E. Kuehni ◽

Jane S. Lucas

Keyword(s):

Secondary Care ◽

Primary Ciliary Dyskinesia ◽

High Speed ◽

Task Force ◽

Diagnostic Testing ◽

List Type ◽

European Respiratory Society ◽

Term Infants ◽

Task Force Report ◽

Ciliary Dyskinesia

Key pointsPrimary ciliary dyskinesia (PCD) is a genetically and clinically heterogeneous disease characterised by abnormal motile ciliary function.There is no “gold standard” diagnostic test for PCD.The European Respiratory Society (ERS) Task Force Guidelines for diagnosing PCD recommend that patients should be referred for diagnostic testing if they have several of the following features: persistent wet cough; situs anomalies; congenital cardiac defects; persistent rhinitis; chronic middle ear disease with or without hearing loss; or a history, in term infants, of neonatal upper and lower respiratory symptoms or neonatal intensive care admission.The ERS Task Force recommends that patients should be investigated in a specialist PCD centre with access to a range of complementary tests: nasal nitric oxide, high-speed video microscopy analysis and transmission electron microscopy. Additional tests including immunofluorescence labelling of ciliary proteins and genetic testing may also help determine the diagnosis.Educational aimsThis article is intended for primary and secondary care physicians interested in primary ciliary dyskinesia (PCD), i.e. those who identify patients for testing, and those involved in diagnosing and managing PCD patients. It aims: to inform readers about the new European Respiratory Society Task Force Guidelines for diagnosing patients with PCDto enable primary and secondary care physicians to: identify patients who need diagnostic testing; understand the diagnostic tests that their patients will undergo, the results of the tests and their limitations; and ensure that appropriate care is subsequently delivered.

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Deep phenotyping, including quantitative ciliary beating parameters, and extensive genotyping in primary ciliary dyskinesia

Journal of Medical Genetics ◽

10.1136/jmedgenet-2019-106424 ◽

2019 ◽

Vol 57 (4) ◽

pp. 237-244 ◽

Cited By ~ 2

Author(s):

Sylvain Blanchon ◽

Marie Legendre ◽

Mathieu Bottier ◽

Aline Tamalet ◽

Guy Montantin ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

High Speed ◽

Genetic Disorder ◽

Beat Frequency ◽

Ciliary Beat Frequency ◽

Central Complex ◽

Ciliary Beating ◽

Recovery Stroke ◽

Biallelic Mutations ◽

Ciliary Dyskinesia

BackgroundPrimary ciliary dyskinesia (PCD) is a rare genetic disorder resulting in abnormal ciliary motility/structure, extremely heterogeneous at genetic and ultrastructural levels. We aimed, in light of extensive genotyping, to identify specific and quantitative ciliary beating anomalies, according to the ultrastructural phenotype.MethodsWe prospectively included 75 patients with PCD exhibiting the main five ultrastructural phenotypes (n=15/group), screened all corresponding PCD genes and measured quantitative beating parameters by high-speed video-microscopy (HSV).ResultsSixty-eight (91%) patients carried biallelic mutations. Combined outer/inner dynein arms (ODA/IDA) defect induces total ciliary immotility, regardless of the gene involved. ODA defect induces a residual beating with dramatically low ciliary beat frequency (CBF) related to increased recovery stroke and pause durations, especially in case of DNAI1 mutations. IDA defect with microtubular disorganisation induces a low percentage of beating cilia with decreased beating angle and, in case of CCDC39 mutations, a relatively conserved mean CBF with a high maximal CBF. Central complex defect induces nearly normal beating parameters, regardless of the gene involved, and a gyrating motion in a minority of ciliated edges, especially in case of RSPH1 mutations. PCD with normal ultrastructure exhibits heterogeneous HSV values, but mostly an increased CBF with an extremely high maximal CBF.ConclusionQuantitative HSV analysis in PCD objectives beating anomalies associated with specific ciliary ultrastructures and genotypes. It represents a promising approach to guide the molecular analyses towards the best candidate gene(s) to be analysed or to assess the pathogenicity of the numerous sequence variants identified by next-generation-sequencing.

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Nasal NO, high-speed video microscopy, electron microscopy, and genetics: a primary ciliary dyskinesia puzzle to complete

Pediatric Research ◽

10.1038/pr.2014.79 ◽

2014 ◽

Vol 76 (3) ◽

pp. 321-321 ◽

Cited By ~ 2

Author(s):

Deborah Snijders ◽

Ilaria Bertozzi ◽

Angelo Barbato

Keyword(s):

Electron Microscopy ◽

Primary Ciliary Dyskinesia ◽

High Speed ◽

Video Microscopy ◽

High Speed Video ◽

Ciliary Dyskinesia

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A Revised Protocol for Culture of Airway Epithelial Cells as a Diagnostic Tool for Primary Ciliary Dyskinesia

Journal of Clinical Medicine ◽

10.3390/jcm9113753 ◽

2020 ◽

Vol 9 (11) ◽

pp. 3753

Author(s):

Janice L. Coles ◽

James Thompson ◽

Katie L. Horton ◽

Robert A. Hirst ◽

Paul Griffin ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Ciliary Dyskinesia ◽

High Speed ◽

Ex Vivo ◽

Cell Damage ◽

Airway Epithelial Cells ◽

University Hospital ◽

Mitigation Measures ◽

Diagnostic Certainty ◽

Ciliary Dyskinesia

Air–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.

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Incidence of Primary Ciliary Dyskinesia in Children with Recurrent Respiratory Diseases

Annals of Otology Rhinology & Laryngology ◽

10.1177/000348949710601008 ◽

1997 ◽

Vol 106 (10) ◽

pp. 854-858 ◽

Cited By ~ 50

Author(s):

André Coste ◽

Marie-Claude Millepied ◽

Catherine Chapelin ◽

Philippe Reinert ◽

Françoise Poron ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Respiratory Tract Infections ◽

Beat Frequency ◽

Ciliary Beat Frequency ◽

Systematic Investigation ◽

Ciliated Cells ◽

Tract Infections ◽

Ciliary Dyskinesia ◽

Ciliary Ultrastructure ◽

Recurrent Respiratory Tract Infections

The goal of the study was to evaluate the incidence of primary ciliary dyskinesia (PCD) in children suffering from recurrent respiratory tract infections (RRIs) by means of a noninvasive method. Respiratory ciliated cells were collected by nasal brushing in 118 children (4.6 ± 2.5 years) with RRIs. The ciliary beat frequency (CBF) was measured with a stroboscopic method, and when the CBF was abnormal, the ciliary ultrastructure was analyzed by a quantitative method. The CBF could be measured in 106 patients (90%) and was abnormal in 15 patients. The ciliary ultrastructure was found to be abnormal in 11 of 15 patients: PCD was diagnosed in 6 cases, and acquired ciliary defects were observed in the remaining 5 patients. Our conclusion, that PCD is rare but not exceptional (5.6%) in children with RRIs, justifies the systematic investigation of ciliated cells in such patients. For this purpose, nasal brushing can be used to sample ciliated cells even in young children.

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Accuracy of diagnostic testing in primary ciliary dyskinesia

European Respiratory Journal ◽

10.1183/13993003.00749-2015 ◽

2015 ◽

Vol 47 (3) ◽

pp. 837-848 ◽

Cited By ~ 52

Author(s):

Claire L. Jackson ◽

Laura Behan ◽

Samuel A. Collins ◽

Patricia M. Goggin ◽

Elizabeth C. Adam ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Primary Ciliary Dyskinesia ◽

High Speed ◽

Diagnostic Testing ◽

Standard Test ◽

Diagnostic Process ◽

Published Data ◽

Transmission Electron ◽

Repeated Sampling ◽

Ciliary Dyskinesia

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.

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Ciliary Feature Counter: A program for the Quantitative Assessment of Cilia to Diagnose Primary Ciliary Dyskinesia

Diagnostics ◽

10.3390/diagnostics10080524 ◽

2020 ◽

Vol 10 (8) ◽

pp. 524

Author(s):

Andreia L. Pinto ◽

Ranjit K. Rai ◽

Claire Hogg ◽

Thomas Burgoyne

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Primary Ciliary Dyskinesia ◽

Quantitative Assessment ◽

Consensus Guideline ◽

International Consensus ◽

Transmission Electron ◽

Speed Up ◽

Ciliary Dyskinesia ◽

Ciliary Ultrastructure

Primary ciliary dyskinesia (PCD) is a disorder that affects motile cilia in the airway that are required for the removal of mucus, debris, and pathogens. It is important to diagnose PCD in early childhood to preserve lung function. The confirmation of a diagnosis relies on the assessment of ciliary ultrastructure by transmission electron microscopy (TEM). TEM involves the quantitative assessment of the ciliary ultrastructure to identify PCD defects as well as abnormalities resulting from infection. Many specialist diagnostic centres still rely on physical counters to tally results and paper notes to summarise findings before transferring the results to computer databases/records. To speed up the diagnostic data collection and increase the protection of patient information, we have developed digital ciliary feature counters that conform to the PCD reporting international consensus guideline. These counters can be used on a computer or tablet, and automatically generate notes regarding sample observations. We show that the digital counters are easy to use and can generate TEM diagnostic reports that will be useful for many PCD diagnostic centres.

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