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Editorial |

Assessment of Ciliary Beat Pattern: Variability in Healthy Control Subjects Has Implications for Use as Test for Primary Ciliary Dyskinesia FREE TO VIEW

Margaret W. Leigh, MD; Michael R. Knowles, MD
Author and Funding Information

FINANCIAL/NONFINANCIAL DISCLOSURES: The authors have reported to CHEST the following: M. W. L. and M. R. K. have received support for primary ciliary dyskinesia (PCD) research from the National Institutes of Health (U54HL096458 funded through a collaboration between the National Center for Advancing Translational Services (NCATS) and the National Heart, Lung, and Blood Institute) for the Genetic Disorders of Mucociliary Clearance Consortium, which is a part of the NCATS Rare Diseases Clinical Research Network (RDCRN). RDCRN is an initiative of the Office of Rare Diseases Research. M. W. L. served on the PCD Advisory Board for Vertex in October 2016.

aMarsico Lung Institute and the Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC

bDepartment of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC

CORRESPONDENCE TO: Margaret W. Leigh, MD, Department of Pediatrics, University of North Carolina at Chapel Hill, 450 MacNider Bldg CB# 7127, Chapel Hill, NC 27599


Copyright 2016, American College of Chest Physicians. All Rights Reserved.


Chest. 2017;151(5):958-959. doi:10.1016/j.chest.2016.11.025
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Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder of ciliary biogenesis, structure, and function. Diagnosis can be challenging. Several diagnostic tests have been used, including assessment of ciliary beat pattern by high-speed videomicroscopy, ciliary ultrastructure by transmission electron microscopy, nasal nitric oxide measurement, and, more recently, immunofluorescence testing using a panel of antibodies and genetic testing using a panel of PCD-associated genes. All of these tests require specialized equipment and expertise for interpretation and none of these tests has the accuracy needed for a “stand alone” diagnostic test.

FOR RELATED ARTICLE SEE PAGE 993

Consensus statements focused on diagnosis and management of PCD have been published by the European Respiratory Society (ERS) task force in 2009 and, more recently, by the North American Research Consortium (Genetic Disorders of Mucociliary Clearance) and the PCD Foundation in 2016. Although both consensus statements recommend a complementary panel of tests and emphasize the need for specialized diagnostic centers, they differ on the relative emphasis on assessment of ciliary beat pattern. The ERS group defined ciliary beat pattern analysis as a key diagnostic technique; subsequently, multiple centers across Europe have adopted this approach. However, the North American group recommended this technique only for those few select centers that are highly experienced with this technology because abnormal beat pattern may be subtle in some patients with PCD (risk for false negatives) and may be associated with non-PCD factors, such as poor quality of the biopsy specimen or recent respiratory illness (risk for false positives). A further concern expressed in the North American statement was that there are “no prospective studies examining inter-rater agreement for functional ciliary analysis,” even though this assessment is qualitative (or semiquantitative if a scoring system is used). In addition, rigorous assessment of ciliary beat pattern can be quite time-consuming. An automated approach for analysis of ciliary beat pattern has been reported, but does not provide a description of the variation in ciliary motion within a sample and has not been tested widely or validated on follow-up clinical samples.

The manuscript by Kempeneers et al in this issue of CHEST uses digital high-speed videomicroscopy (DHSV) to evaluate variation in ciliary beat pattern in 3 different beating planes (profiles) in ciliary brush biopsies from healthy subjects. The major strengths of this study are the methodologies and rigorous quantitative approach (see the detailed Methods section in the article’s Appendix) that include well-defined criteria for selection of subjects, detailed protocol for acquisition and processing of biopsies, well-defined criteria for selection of “undisrupted ciliary edges,” and the use of independent observers for qualitative and semiquantitative assessment of ciliary beat pattern. Despite the carefully controlled selection of healthy subjects and ciliary edges, only 2/13 subjects had completely normal ciliary beat pattern in the 3 profiles evaluated, and only 69.1% of the edges evaluated in the sideways profile had a uniform ciliary beat pattern. Some regions within the ciliary edges had immotile, stiff, or asynchronous ciliary beat patterns. This lack of uniformity of ciliary beat pattern in carefully selected epithelial edges from healthy control subjects emphasizes the need for further quantitative standardization and prospective, blinded, diagnostic studies of patients with PCD with a range of genetic and ultrastructural defects in cilia before this test can be validated and extended beyond highly specialized centers. As emphasized by the authors, this variability has implications for attempts to develop automated methods for assessment of ciliary beat pattern, because these automated approaches have a limited region of interest that may not be representative for the whole sample. Furthermore, these automated approaches will require standardization and blinded studies of samples from patients with PCD before the samples can be used in the clinical arena for diagnostic purposes.

Analysis of ciliary beat pattern in groups of patients with PCD with a defined ultrastructural defect and/or with biallelic mutations in a PCD-associated gene have identified some distinct alterations in ciliary beat pattern in PCD. Patients with outer dynein arm defects or with outer and inner dynein arm defects have a high proportion of immotile cilia (55% and 80%, respectively) and have a stiff beat pattern in motile cilia. In contrast, patients with a central apparatus defect with transposition of a peripheral doublet into the center have no immotile cilia, but their cilia beat in a circular pattern. Patients with biallelic mutations in DNAH11 have normal ultrastructure and a hyperkinetic ciliary beat with a subtle stiffness to the bend of the cilia. It is not clear whether these subtle defects would be recognized as different from normal without prior knowledge of the genetic defect; hence, there is a risk for false negatives using DHSV for diagnostic purposes. Further, some nonspecific secondary changes in ciliated epithelium, such as damage by inflammation and viral infections, are associated with ciliary dyskinesia that may be falsely identified as PCD,,; therefore, biopsies should be taken after full recovery from acute respiratory illnesses and other inflammatory insults to decrease likelihood of false positives. Despite screening for respiratory illnesses in the 6 weeks before biopsy, a study from the UK National Diagnostic Centres for PCD showed that repeat biopsy had normal ultrastructure in 5/21 patients with an inner dynein arm defect on initial biopsy and in 1/6 patients with a radial spoke defect on the initial biopsy. Of note, all 6 individuals with normal ultrastructure on repeat biopsy had abnormal ciliary beat pattern on the initial biopsy, but normal ciliary beat pattern on the repeat biopsy. Therefore, repeat biopsies with DHSV assessment studies may be needed, but the appropriate number or timing of repeat testing has not been defined.

In summary, this rigorous and quantitative study by Kempeneers et al represents the type of systematic approach that is needed to standardize assessment of ciliary beat pattern as a diagnostic method in PCD. The most recent ERS guidelines for PCD diagnosis from November 2016 made only a “weak recommendation” for use of high-speed videomicroscopy analysis because “there is no consensus on appropriate cell processing and method of ciliary assessment” and “standardised protocols and thresholds need to be developed.” We look forward to further standardization of ciliary beat pattern assessment that includes the same rigor as the study by Kempeneers et al and systematic blinded studies of cilia from patients with PCD, which will allow better discrimination of subtle PCD changes from normal, when performed by well-trained and experienced investigators.

References

Barbato A. .Frischer T. .Kuehni C.E. .et al Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children. Eur Respir J. 2009;34:1264-1276 [PubMed]journal. [CrossRef] [PubMed]
 
Shapiro A.J. .Zariwala M.A. .Ferkol T. .et al Diagnosis, monitoring, and treatment of primary ciliary dyskinesia: PCD Foundation consensus recommendations based on state of the art review. Pediatr Pulmonol. 2016;51:115-132 [PubMed]journal. [CrossRef] [PubMed]
 
Quinn S.P. .Zahid M.J. .Durkin J.R. .Francis R.J. .Lo C.W. .Chennubhotla S.C. . Automated identification of abnormal respiratory ciliary motion in nasal biopsies. Sci Transl Med. 2015;7:299ra124- [PubMed]journal. [CrossRef] [PubMed]
 
Kempeneers C. .Seaton C. .Chilvers M.A. . Variation in ciliary beat pattern in three different beating planes in healthy subjects. Chest. 2017;151:993-1001 [PubMed]journal
 
Chilvers M.A. .Rutman A. .O'Callaghan C. . Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia. J Allergy Clin Immunol. 2003;112:518-524 [PubMed]journal. [CrossRef] [PubMed]
 
Raidt J. .Wallmeier J. .Hjeij R. .et al Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia. Eur Respir J. 2014;44:1579-1588 [PubMed]journal. [CrossRef] [PubMed]
 
Smith C.M. .Kulkarni H. .Radhakrishnan P. .et al Ciliary dyskinesia is an early feature of respiratory syncytial virus infection. Eur Respir J. 2014;43:485-496 [PubMed]journal. [CrossRef] [PubMed]
 
Chilvers M.A. .McKean M. .Rutman A. .Myint B.S. .Silverman M. .O'Callaghan C. . The effects of coronavirus on human nasal ciliated respiratory epithelium. Eur Respir J. 2001;18:965-970 [PubMed]journal. [CrossRef] [PubMed]
 
Thomas B. .Rutman A. .Hirst R.A. .et al Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma. J Allergy Clin Immunol. 2010;126:722-729 [PubMed]journal. [CrossRef] [PubMed]
 
O'Callaghan C. .Rutman A. .Williams G.M. .Hirst R.A. . Inner dynein arm defects causing primary ciliary dyskinesia: repeat testing required. Eur Respir J. 2011;38:603-637 [PubMed]journal. [CrossRef] [PubMed]
 
Lucas JS, Barbato A, Collins SA, et al. European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia [published online ahead of print November 11, 2016].Eur Respir J.http://dx.doi.org/10.1183/13993003.01090-2016.
 

Figures

Tables

References

Barbato A. .Frischer T. .Kuehni C.E. .et al Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children. Eur Respir J. 2009;34:1264-1276 [PubMed]journal. [CrossRef] [PubMed]
 
Shapiro A.J. .Zariwala M.A. .Ferkol T. .et al Diagnosis, monitoring, and treatment of primary ciliary dyskinesia: PCD Foundation consensus recommendations based on state of the art review. Pediatr Pulmonol. 2016;51:115-132 [PubMed]journal. [CrossRef] [PubMed]
 
Quinn S.P. .Zahid M.J. .Durkin J.R. .Francis R.J. .Lo C.W. .Chennubhotla S.C. . Automated identification of abnormal respiratory ciliary motion in nasal biopsies. Sci Transl Med. 2015;7:299ra124- [PubMed]journal. [CrossRef] [PubMed]
 
Kempeneers C. .Seaton C. .Chilvers M.A. . Variation in ciliary beat pattern in three different beating planes in healthy subjects. Chest. 2017;151:993-1001 [PubMed]journal
 
Chilvers M.A. .Rutman A. .O'Callaghan C. . Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia. J Allergy Clin Immunol. 2003;112:518-524 [PubMed]journal. [CrossRef] [PubMed]
 
Raidt J. .Wallmeier J. .Hjeij R. .et al Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia. Eur Respir J. 2014;44:1579-1588 [PubMed]journal. [CrossRef] [PubMed]
 
Smith C.M. .Kulkarni H. .Radhakrishnan P. .et al Ciliary dyskinesia is an early feature of respiratory syncytial virus infection. Eur Respir J. 2014;43:485-496 [PubMed]journal. [CrossRef] [PubMed]
 
Chilvers M.A. .McKean M. .Rutman A. .Myint B.S. .Silverman M. .O'Callaghan C. . The effects of coronavirus on human nasal ciliated respiratory epithelium. Eur Respir J. 2001;18:965-970 [PubMed]journal. [CrossRef] [PubMed]
 
Thomas B. .Rutman A. .Hirst R.A. .et al Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma. J Allergy Clin Immunol. 2010;126:722-729 [PubMed]journal. [CrossRef] [PubMed]
 
O'Callaghan C. .Rutman A. .Williams G.M. .Hirst R.A. . Inner dynein arm defects causing primary ciliary dyskinesia: repeat testing required. Eur Respir J. 2011;38:603-637 [PubMed]journal. [CrossRef] [PubMed]
 
Lucas JS, Barbato A, Collins SA, et al. European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia [published online ahead of print November 11, 2016].Eur Respir J.http://dx.doi.org/10.1183/13993003.01090-2016.
 
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