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Abstract :
[en] Primary ciliary dyskinesia (PCD) is a rare inherited motile ciliopathy in which respiratory cilia beat abnormally, leading to impaired mucociliary clearance. PCD is rare but the exact
prevalence remains unknown, as current literature shows that PCD is under-diagnosed and diagnosed late in Europe. One reason about the delay in PCD diagnosis is that there is no "gold standard" test for PCD, the diagnosis requiring a panel of different diagnostic tests. Ciliary videomicroscopy, using digital high speed videomicroscopy (DHSV), is one of the PCD diagnostic tests, allowing the visualization and the evaluation of respiratory ciliary beating. Previous studies described that ciliary beating evaluation using DHSV is a good discriminating test for PCD. However, the European Respiratory Society guidelines do not recognize ciliary videomicroscopy to confirm a PCD diagnosis in isolation, and the American Thoracic Society guidelines do not include ciliary videomicroscopy in the diagnostic algorithm, due to two persistent issues. The first issue is the lack of an international standardized protocol for ciliary videomicroscopy. Indeed, the use of different protocols may lead to different reference values for ciliary function between the different PCD diagnostic centres, because different ways of performing each step of the ciliary videomicroscopy technique may have an effect on ciliary beating. The second issue is the difficulty to define the accurate sensitivity and specificity of ciliary videomicroscopy, as there is no gold standard reference test. Previous studies evaluated the ability of ciliary videomicroscopy to diagnose PCD, but this might be inaccurate, as it has been evaluated against a non-optimal reference standard (incomplete, or including ciliary videomicroscopy). In the first part of this work, we identified the different steps of the ciliary videomicroscopy protocol that differ between centres, and that may have an effect on ciliary beating. We highlighted three methodological steps differing between centres (1) the storage duration and the storage temperature of the respiratory ciliated samples before ciliary beating video recording, (2) the medium used to store the respiratory ciliated samples before ciliary beating video recording and (3) the temperature used during ciliary beating video recording. In
addition, as this project had to be carried out during the Covid-19 pandemic, we had to adapt the ciliary videomicroscopy protocol used in our laboratory in order to continue the
experiments using live respiratory samples that might be contaminated. In the second part of our work, we studied the effects of the variations of these three methodological steps on ciliary beating. We demonstrated that the storage temperature of the samples before video recording (4°C or room temperature (RT)) and the temperature during video recording (RT or 37°C), modify ciliary beating, and need to be taken into account when establishing normative data for ciliary function, as well as when establishing a future
standardised protocol. However, our results showed that a storage duration up to 9 hours,
and the medium used to store ciliated samples before video recording (M199 or RPMI 1640), do not modify ciliary beating, and might vary when defining normative data, or when establishing a standardized protocol. The third part of this work focused on the ability of ciliary videomicroscopy to diagnose PCD, using a complete reference standard based on all available diagnostic tests without including the results of ciliary videomicroscopy evaluated from fresh respiratory samples. Our results showed that ciliary videomicroscopy, performed at RT or at 37°C, was a good discriminating test for PCD. Finally, the last part of this project was to develop a method to enable the use of ciliary videomicroscopy as a diagnostic tool for PCD in dogs. We found that nasal turbinate brushings and bronchial brushings, performed after nasal or bronchial lavage, were the optimal sampling methods to obtain respiratory ciliated cells from dogs. In addition, normal reference values of ciliary function were established and were used to diagnose a dog with a suspicion of PCD. In conclusion, until now, ciliary videomicroscopy is a highly sensitive and specific test for PCD diagnosis, but is not recognized/not included in the international PCD diagnostic guidelines, as the test lacks standardization, and its accuracy to diagnose PCD has been evaluated against non-optimal reference standards. We found that ciliary videomicroscopy was a good discriminating test for PCD, when this was calculated against an optimal reference standard and we identified some methodological steps in the videomicroscopy protocol that might modify ciliary beating (the storage temperature, and the temperature during video recording). The identification of the different steps of the ciliary videomicroscopy protocol that need to be standardized, as their modification induce changes in ciliary beating, is a crucial step in the development of a future international standardised operating procedure.
