Editorial

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous autosomal-recessive disorder. It is the most frequent type of ciliopathy caused by congenital abnormalities in the structure and or function of cilia. The incidence of PCD is estimated to be between 1:10,000 and 1:20,000 live births¹ and causes severe, chronic destructive airway disease with progressive loss of lung function².

Historically, clinical manifestations of PCD were described in the early 1900s, but it was Manus Kartagener who first noticed the typical association of the triad of symptoms, comprising bronchiectasis, situs inversus, and sinusitis in 1933 and named the disorder after his name, as Kartagener’s syndrome³. Much later, Afzelius using transmission electron microscopy (TEM) identified an absence of dynein arms in the axoneme of respiratory cilia and sperm flagella, in patients with Kartagener’s syndrome⁴. This landmark discovery, sparked off a flurry of investigations, highlighting the critical role of TEM in the diagnosis of PCD^5,6. As a result, TEM was established as the “gold standard” method for diagnosing PCD, a concept that continued to be considered as valid till the early 1990s⁷. Currently, the diagnosis of PCD remains challenging, despite the fact that a number of newer techniques have been developed and admittedly have increased the sensitivity and accuracy of diagnosis of PCD patients. These techniques include beyond TEM, the assessment of ciliary beat frequency and pattern, the measurement of nasal nitric oxide (nNO) levels, molecular genetics as well as immunofluorescence analysis⁸.

The overturn in PCD diagnostics was caused by recent findings of genetic studies, which demonstrated that a substantial subset of PCD patients exhibits normal ultrastructure and cannot be identified by TEM analysis^9–11. As a result, current recommendations changed and require the performance of additional diagnostic investigations such as high-speed video microscopy, nNO, immunofluorescence, and genetic analysis¹². This multidisciplinary approach for the establishment of PCD diagnosis, involving genetic, ciliary motility, and immunofluorescence studies, provides novel insights into the underlying mechanisms and ultrastructural pathology of PCD that merited an appraisal in a special issue of UP. Furthermore, ultrastructural defects previously identified by TEM in the investigation of ciliary disorders but with questionable diagnostic significance, such as inner dynein arm defects¹³ had to be discussed and placed in context. A special issue would also provides the opportunity to present technological advances in laboratory, or digital techniques, aiming to improve the resolution of traditional TEM micrographs, as well as novel research applications, such as electron tomography, that have been shown to be useful in the assessment of ciliary ultrastructure¹⁴. Of additional interest is the contribution of TEM to the assessment of motile cilia defects, in conditions other than PCD, e.g., the Bardet Biedl¹⁵ syndrome and Leber Congenital Amaurosis¹⁶.

Overall, ultrastructural pathologists need to reevaluate the contribution of TEM analysis in the diagnostic work-up for PCD and at the same be updated on the emerging electron microscopy based, research applications which advance our understanding on the pathogenesis of ciliary disorders. In addition, the wider audience of clinicians and scientists, involved in PCD, would benefit from the presentation of the current evidence on the role and limitations of TEM in the state of the art diagnostic procedures, and research investigations available for diagnosing PCD. The current special tissue meets the above needs and features an editorial and six manuscripts from experienced clinicians and scientists who have worldwide experience in the diagnosis of PCD. The first article by Shapiro and Leigh titled: “The Value of Transmission Electron Microscopy for Primary Ciliary Dyskinesia Diagnosis in the Era of Molecular Medicine: Genetic Defects with Normal and Non-Diagnostic Ciliary Ultrastructure,” sets the scene and “hits the nail on the head,” as it presents an appraisal of the contemporary diagnostic value of TEM in PCD diagnosis. It presents the evidence which changed TEM’s role from traditionally been the gold standard method, to its current diagnostic capabilities in the era of molecular medicine. It highlights the importance of using a multidisciplinary approach as a must, that enhances diagnostic accuracy for PCD and provides emerging data from genetic studies which define many genetic defects, that have been shown to be associated with a normal ultrastructure. The second manuscript by Werner and Kouis extrapolates the discussion further and provides a contemporary insight into the core question on the current usefulness of TEM under the title “Should TEM and ultrastructural cilia evaluation remain part of the diagnostic work-up of PCD?”. This manuscript has been structured into a pros and cons format in order to provide the reader with a targeted and an up-to-date knowledge of both sets of arguments, relating to the contemporary use of TEM in PCD diagnosis.

The third manuscript titled: “Secondary TEM defects in PCD diagnostics” by Shoemark and Dixon, discusses the very important aspect of secondary defects, the presence of which often complicates the accurate diagnosis of PCD. Since TEM still remains a useful diagnostic tool for PCD diagnosis, it is of paramount importance for the scientific PCD community to be fully aware of the diagnostic pitfalls that can be generated by secondary defects, which are very frequent in the respiratory epithelium samples examined, as most of the referred patients for diagnostic work-up suffer from chronic respiratory infections. Manuscript number four titled: “Application of laboratory and digital techniques for visual enhancement during the ultrastructural assessment of abnormal cilia” by Schroeder, presents a hybrid of old and new techniques that are currently in use for the enhancement of cilia ultrastructure imaging. The main aim of these techniques is to facilitate the more accurate assessment of the axoneme ultrastructure in order to enable the detection of PCD defects, such as the loss of dynein arms. In particular, the described computer-enhanced method may be useful in borderline cases of PCD, where the ultrastructure of numerous axonemes, sometimes more than 100, is required in order to increase diagnostic accuracy.

Similarly manuscript number five titled: “Applications of emerging TEM technologies in PCD research and diagnosis” by Shoemark, highlights the importance of using electron tomography, an accessory attached to the normal TEM, that is widely available for detecting defects in the axoneme of PCD patients, which cannot be detected by routine TEM. These important emerging applications give additional value to the use of TEM, making the future use of TEM in PCD research even more important. It is expected that more new PCD causative genetic defects will be identified, using molecular genetics. Therefore better imaging will facilitate the correlation of these genetic defects with newly identified ultrastructural abnormalities.

The final manuscript number six, titled: “Motile cilia defects in diseases other than Primary Ciliary Dyskinesia: The contemporary diagnostic and research role for Transmission Electron Microscopy,” by Mitchison and Shoemark, provides an excellent account of the contemporary use of TEM in diagnosing defects in motile cilia, in an emerging family of diseases including syndromes, other than PCD. Indeed, the use of TEM and the more specialized techniques such as cryo-EM and electron tomography are providing unique insights into defects in cilia, critical for diagnosis and research and for correlating genotypes with phenotypes.

In summary, we believe that this special issue on ciliary disorders, with a focus on PCD, presents a timely appraisal of new developments in the field and provides comprehensive and updated information on the current diagnostic and research modalities that are being applied for the diagnosis of PCD and other ciliary disorders. Centrally positioned in the diagnostic modalities required for PCD diagnosis is TEM, which remains a very powerful tool, despite its limitations and the development of new diagnostic techniques. We hope that this edition will be read by many and that it will serve as a reference document of interest to both clinicians and scientists, who are either new in the field or are already working in PCD diagnosis and/or research.