Rapid diagnosis of primary ciliary dyskinesia: cell culture and soft computing analysis

Abstract

Diagnosis of primary ciliary dyskinesia (PCD) sometimes requires repeated nasal brushing to exclude secondary ciliary alterations. Our aim was to evaluate whether the use of a new method of nasal epithelial cell culture can speed PCD diagnosis in doubtful cases and to identify which are the most informative parameters by means of a multilayer artificial neural network (ANN).A cross-sectional study was performed in patients with suspected PCD. All patients underwent nasal brushing for ciliary motion analysis, ultrastructural assessment and evaluation of ciliary function after ciliogenesis in culture by ANN.151 subjects were studied. A diagnostic suspension cell culture was obtained in 117 nasal brushings. A diagnosis of PCD was made in 36 subjects (29 of whom were children). In nine out of the 36 patients the diagnosis was made only after a second brushing, because of equivocal results of both tests at first examination. In each of these subjects diagnosis of PCD was confirmed by cell culture results.Cell culture in suspension evaluated by means of ANN allows the separation of PCD from secondary ciliary dyskinesia patients after only 5 days of culture and allows diagnosis to be reached in doubtful cases, thus avoiding the necessity of a second sample.

Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure

“…PCD was defined as the absence of coordinated ciliary activity after ciliogenesis in culture, indicating that efficient ciliary transport was absent (see Additional files 1, 2 and 3 for examples of normal and abnormal coordination of ciliary activity). The validity of this evaluation of coordination to distinguish PCD from SCD was replicated by an independent group [31]. …”

Gene editing ofDNAH11restores normal cilia motility in primary ciliary dyskinesia

“…Pseudotyping vectors with filovirus, baculovirus and influenza coat proteins and pretreating the respiratory mucosa with clinically approved mucolytic agents44–46 should overcome local barriers and allow meaningful HR levels to be obtained in vivo. (2) Spheroids were transduced at day 6 post-collection, and normalisation of ciliary motility was observed by 2 days later, at a time when spheroids still retain the original cilia 6. Although little is known about the time required for assembly or turnover of axonemal proteins in mammalian systems,12 the observation that the original cilia are maintained in culture for about 2 weeks and are then progressively replaced by new cilia6 20 suggests that, in the present work, normalisation of ciliary motility occurred by replacement of defective with normal DNAH11 protein.…”

“…In culture, ciliated epithelial cells form spheroids consisting of layers of respiratory epithelial cells with ciliated apical membranes orientated outward which, in the presence of normal ciliary motility, but not in patients with PCD, move and rotate in the medium. Spheroids maintain their cilia for 14–20 days of cultivation and are usually identifiable after 24–48 h of culture 6 20 21. It has been speculated that, after correction of a congenital defect or as observed in secondary ciliary dyskinesia, restoration of normal ciliary activity is rapid and does not require formation of new cilia, which takes 2–3 weeks in vitro 6 12 20…”

“…Videos before and after cell manipulation were acquired at 37°C by positioning the well culture plates on a heated plate under the microscope; analyses were performed by two investigators (MPif and MPir, who were blinded to cell manipulation) as described previously 6. Approximately 120 spheroids were treated from each patient.…”

Is the sensitivity of primary ciliary dyskinesia detection by ciliary function analysis 100%?

“…We wish to thank M. Boon and co-workers for their careful reading of our manuscript and to apologise to them and the readers for our mistake in the preparation of table 2 of our original article [1]. We erroneously reversed the sensitivity and specificity values, as well as the negative predictive value and positive predictive value for the first four parameters (rotation of spheroids, migration of spheroids, ability of cilia to remove debris, and normal ciliary beat pattern).…”