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

RESULTS

151 subjects were studied (fig. 1). A diagnostically useful suspension cell culture was obtained from nasal brushing samples in 117 (77.5%; 65 males, 91 children, age range 1.5 months to 57.0 years; median age (IQR) 9.30 (12.0) years) out of 151 subjects (all details are shown in online supplementary table S1). In 23 (15.2%) patients the cultures became infected during the first 48 h and in 11 (7.3%) subjects the culture was unsuccessful, either because of a lack of cells in the brushing sample, or because no cilia could be found in spheroids throughout the period of culture. Moreover, because only a small sample could be obtained, the duration of cell culture in suspension, usually 20 days, was reduced to 10 and 15 days, respectively, in nine (7.7%) and 10 (8.5%) out of 117 patients.

Ciliary motion analysis (abnormal motion patterns, including immotile cilia and/or very low ciliary beat frequency) and TEM evaluation of cilia (alterations of the central pair and deficiencies of the dynein arms, associated with a small proportion of swollen cilia and compound cilia) allowed the eventual definitive diagnosis of PCD in 36 (30.8%) subjects (29 children). In nine out of the 36 patients, the diagnosis was obtained only after a second brushing because of equivocal results in one subject or the presence of normal ciliary ultrastructure with nonflexible and hyperkinetic ciliary motion pattern in eight subjects at the first examination (all details shown in online supplementary table S2). In each of these nine subjects, the diagnosis of PCD was strongly suggested prior to any TEM results being obtained by the results of cell cultures (table 1) (online supplementary video clip 2).

In the remaining 81 (69.2%) patients (68 children), ciliary motion analysis demonstrated abnormal patterns in a small proportion of cilia, prevalence of thick cilia and low ciliary beat frequency, compatible with SCD (secondary changes were found in one patient only after the second brushing after intensive and prolonged treatment with antibiotics, but SCD was correctly identified from the culture). In these 81 subjects, TEM evaluation clearly showed nonspecific abnormalities compatible with chronic inflammation (prevalence of swollen cilia and compound cilia). In all these patients the diagnosis of SCD was robustly suggested by the prior results of cell cultures.

Moreover, as can be seen in table 1, ciliary activity evaluation, over the whole 20-day period of suspension cell culture, demonstrated a rotation of the spheroids in 64 out of 81 (79%) subjects with final diagnosis of SCD, and only in one (2.8%) out of 36 patients with PCD (rotation of one spheroid 10 days after the start of the cell cultures). In this patient, the diagnosis was confirmed by DNAH11 compound heterozygous mutations [11]. Migration of the spheroids was found in 14 (17.3%) out of 81 subjects with SCD, but in no PCD patients, and an ability of cilia to remove debris in six (7.4%) out of 81 subjects with SCD and in none with PCD. When continuous movements of cilia were detected but migration or rotation of the spheroids with an ability of cilia to remove debris were not observed, a normal ciliary beat pattern was observed at CMA in 52 (64.2%) out of 81 subjects with SCD, but in no patient with PCD. By contrast, in 12 (33.3%) out of 36 of these PCD patients, but in none with SCD, a pathological ciliary pattern was observed (table 1). In particular, a reduced ciliary beat frequency with a markedly restricted beat amplitude and a stiff ciliary motion pattern or an abnormal nonflexible beating pattern with a hyperkinetic beat were detected in four and eight subjects, respectively.

The sensitivity, specificity, PPVs and NPVs of the different parameters for the diagnosis of PCD are reported in table 2.

Taking into consideration the rotation of the spheroids, migration of the spheroids, ability of cilia to remove debris and type of ciliary motion in the range 5–20 days, the ANN-based model was able to identify the final diagnosis in almost all cases (PCD 99.5±0.3%; SCD 98.8±1.2%).

Moreover, since the best combination of sensitivity and specificity was demonstrated by rotation of the spheroids, the ability to correctly distinguish the PCD from SCD by the ANN-based model, taking into account this parameter together with each of the other three, was analysed.

As reported in table 3, the model was able to correctly identify the PCD or SCD in nearly all cases when the rotation of the spheroids and the type of ciliary motion were considered. This was true from day 5 of cell culture. Other combinations were less accurate.

DISCUSSION

The principal finding of the study was that culture in suspension of cells obtained by nasal brushing is possible in around 80% of the samples, and in our hands, using ANNs, this allowed the differentiation of PCD from SCD within 1 week, and well before TEM results become available.

Strengths of the study include the objective analysis of the data using ANNs, i.e. by mathematical algorithms generated by computer. With this approach, we have been able to identify the two parameters, which, when combined, are most diagnostically accurate (rotation of the spheroids together with type of ciliary motion). These have been used to accurately identify nearly 95% of PCD patients after only 5 days of culture. ANNs learn from standard data and capture the knowledge contained in the data. Further strengths include the relatively atraumatic nature of the brushing compared to a forceps biopsy. Originally, cells for culture were obtained with more traumatic methods, such as punch biopsy, which may cause pain and bleeding. Punch biopsy also has the disadvantage of requiring a longer time to establish cell culture compared with cells collected by brushing [7, 13, 14]. The technique does, however, have the advantage of a high yield of culture results. Ciliated air–liquid cultures from both punch and brush biopsies [14] result in the culture of sufficient ciliated tissue to allow electron microscopy of cilia, which is not possible with our simplified technique, but the success rate of this technique is only 54% [14] not 80% as here. However, with the air–liquid technique, the culture success rate decreases to 26% in patients with more severe secondary changes, that is, in the very group in whom the risk of a false-positive diagnosis is more likely, and the cells previously had to be cultured for 21 days, increasing the risk of failure [7]. This is exacerbated by the paucity of the starting material, which tends to reduce further in subsequent passages. However, this present study clearly demonstrates that prolonged culture is not necessary to make the diagnosis of PCD.

The major weakness of the study is that TEM is not possible with this technique, but the PPVs of the different ciliary motion patterns resulted in >98.5% accuracy, thus allowing the diagnosis in almost all patients. Of course, TEM, when diagnostic (for example, outer dynein arm deficiency), is an important test, but it is not the gold-standard, since cases of PCD with normal ultrastructure are well described [5, 11]. This was the case in eight of our patients, who were correctly identified from the beginning by the study of ciliogenesis in culture. Even though we cannot perform electron microscopy on cells cultured by our simplified technique, soft computing allowed us to diagnose PCD before the second brushing and without TEM being available in an additional 22% of the patients within less than a week of the first nasal brushing, without recourse to a second brushing. The result is surprising, given that spheroids of epithelial cells in suspension culture lost cilia during the first week and developed new cilia only after a further 1–2 weeks [15]. We speculate that restoration of normal ciliary activity, in the absence of a congenital defect, does not require the formation of complete new cilia but may occur as a consequence of a normalisation of the normal trafficking of protein complexes between the cell basal body and the cilia tip as required for the assembly, maintenance and function of cilia [16]. In this context, it is worth noting that a second ciliary gene mutation in cis may correct the abnormal phenotype caused by the first, curiously sometimes without apparently restoring the missing substructure [17–20]. We suggest that the integration in a dedicated system of the optical acquisition and the soft computing algorithm will allow the evaluation of time-dependent cell culture results to be gained rapidly, resulting in a cost saving. TEM will thus become a confirmatory tool, given the long delay in obtaining results.

Ideally, we would validate these findings in a second cohort of patients, but it would take a considerable time to generate a sufficiently large number of new patients. However, the network was validated according to a n-fold cross-validation method. Cross-validation is one of several approaches for estimating the performance of a model on unseen data. In a n-fold cross-validation, the original dataset is randomly partitioned (split) into n subsets. For each cross-validation step, a single subset is retained as the validation set (or test set, i.e. validation cohort), and the remaining n-1 sub-subsets are used as training set. The cross-validation process is then repeated n times, with each of the n subsets used exactly once as the test set. The n results from the folds then can be averaged (or otherwise combined) to produce a single estimation. In our work, a five-fold cross-validation process of the different cell culture features at days 5, 10, 15 and 20 was applied; each fold consisted of randomly selected data, and at least one for each category index was included in each fold. We consider that this cross-validation is very robust; much more so than merely splitting the cohort in half using one half as a discovery cohort and one for validation.

The implication of our study is that PCD is rare and difficult to diagnose, and may require repeated testing in doubtful cases [21, 22]. Problems include, first, that TEM changes may be secondary to environmental mucosal injuries, such as inflammation and bacterial or viral infections, allergies and smoking [23], or may result from inappropriate preparation of a specimen [24]; second, in a few patients with immotile cilia, ciliary ultrastructure may be normal or near normal [25–28]. In this regard, the complexities of determining the gold-standard diagnosis have recently been discussed [4]. We believe this new technique will make diagnosis in doubtful cases significantly easier.

If there is diagnostic doubt, identification of the causative genetic mutations would be ideal, as in our very atypical PCD patient with rotation of spheroids, but PCD is a genetically heterogeneous disease and more than 250 potential candidate genes have been identified [29]. Furthermore, genetic studies have the problem of distinguishing disease-producing mutations from harmless polymorphisms. Other investigations such as nasal nitric oxide are part of the diagnostic process [30], but may be normal in rare patients with PCD and low in subjects with other diseases such as upper airway infections and cystic fibrosis [31]. Under these challenging circumstances, culture of ciliated cells is very helpful, since secondary changes are virtually absent after regrowth of the ciliated epithelium in culture while primary defects remain unchanged [32, 33].

In conclusion, the use of a simple culture technique together with the use of ANNs [34] offers new opportunities for the reliable differentiation of PCD from secondary ciliary dyskinesia.