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The primary abnormality in PCD is one of dysmotile cilia as described above. In humans this leads to delayed mucociliary clearance within the respiratory tract and this is responsible for the symptoms of recurrent sinopulmonary infections and subsequent bronchiectasis. Dysmotile cilia can also be demonstrated within the genitourinary tract affecting the fallopian tubes in women and the vas

deferens in men. Both oligospermia and azoospermia have been reported in infertile males with PCD (Pedersen and Rebbe 1975). Identical ultrastructural abnormalities to those seen in cilia may also be seen within the axoneme of the spermatozoal flagella resulting in immotile or hypomotile sperm (Eliasson, Mossberg et al. 1977) (Munro, Currie et al. 1994). These additional sites of ciliary abnormality explain the other main feature of PCD that is subfertility. It is important to be aware that male infertility is not universal and seminal analysis

should be performed in adult males to assess individual fertility (Munro, Currie et al. 1994). Females with PCD are known to have reduced fertility when compared to the general population.

The ependymal cells of the brain are also known to be ciliated but hydrocephalus is not a well documented feature of the human disease.

Unexplained hydrocephalus (Greenstone, Jones et al. 1984) (De-Santi, Magni et al. 1989) has been associated with PCD in rare cases but screening for minor differences in the size of the cerebral ventricles using cerebral imaging

techniques has not been undertaken.

The association of situs inversus in 50% of patients with the above features has previously been discussed and often provides the initial clue to the diagnosis when present. Within the same family siblings may have PCD with or without situs inversus.

The primary symptoms of PCD are present from birth (Whitelaw, Evans et al. 1981) but there is significant variation in both the severity of symptoms and the age at which the condition is diagnosed (Turner, Corkey et al. 1981). In the neonatal period these symptoms include unexplained tachypnoea and pulmonary collapse or consolidation without an obvious predisposing cause. Screening for PCD should also be considered in infants with situs inversus or more complex congenital anomalies such as congenital heart defects,

oesophageal atresia or biliary atresia. Older children may have persistent rhinitis, cough and wheeze, recurrent episodes of upper and lower respiratory tract infection, sinusitis and severe serous otitis media with an associated conductive hearing impairment or ottorhoea following grommet insertion. The diagnosis should also be considered in children with bronchiectasis, atypical asthma and siblings of index cases. In some cases the disease may not be diagnosed until adulthood when the patient presents because of reduced fertility or in the case of a female patient ectopic pregnancy. The symptoms and signs of

PCD at different ages are summarised in Table 3.1. Patients with PCD may therefore present to the GP, paediatrician, adult physician, ENT surgeon, gynaecologist or urologist.

Table 3.5-1: Symptoms of PCD at different ages

Neonate Child Adult

Unexplained tachypnoea Atypical asthma Male Infertility Neonatal pneumonia Chronic productive cough Female subfertility Situs Inversus Bronchiectasis Ectopic pregnancy Affected sibling Chronic severe otitis media

with otorrhoea

Bronchiectasis

Rhinoslnusltls Affected sibling

Rarely: Rarely:

Complex heart disease Oesophageal reflux Oesophageal atresia Nasal polyps Severe oesophageal reflux

Biliary atresia Hydrocephalus

Table 3.5-1: This table summarises the symptoms and signs of PCD at different ages.

The phenotype of PCD shares similarities with other chronic respiratory diseases including cystic fibrosis and immunodeficiency disorders. Patients have been described in which cystic fibrosis and Kartagener syndrome were thought to co­ exist (Burnell and Robertson 1974) (Brown and Smith 1957). Although

theoretically possible it is more likely that cystic fibrosis was associated with situs inversus in these patients as formal ciliary studies were not performed.

Initial investigations will vary depending on the age of presentation but usually includes a plain radiograph of the chest, sputum samples for virology and bacteriology cultures, pulse oximetry, lung function tests, immunoglobulin

analysis with subclasses and a sweat test or cystic fibrosis genotype. Further investigations such as echocardiography, high resolution thin section computed tomography of the chest, serology for mycoplasma and adenovirus, alpha 1 antitrypsin genotype, autoantibody screen, pH studies, immune function studies and fibreoptic bronchoscopy should also be performed where indicated (Bush, Cole et al. 1998).

The definitive diagnosis of PCD depends on the demonstration of impaired mucociliary clearance in association with a primary abnormality of cilia. These primary abnormalities continue to be described although abnormally long cilia (Niggemann, Müller et al. 1992), abnormalities of the basal bodies (Lungarella, De-Santi et al. 1985) and ciliary aplasia are well documented. In most cases the cilia are immotile or dysmotile and the specific ultrastructural abnormality can also be determined. The main primary ciliary defects and ultrastructural abnormalities demonstrated in PCD are summarised in table 3.5-2.

Table 3.5-2: Ultrastructural phenotypes

Dynein Arms

Absent or reduced inner and outer dynein arms Absent or reduced outer dynein arms

Absent or reduced inner dynein arms

Radial spokes

Absent radial spokes/absent inner dynein arms

Microtubules

Absent central pair with transposition of peripheral microtubule doublet to centre

Peripheral microtubule defect

Complete ciliary aplasia Defect of orientation

An electron micrograph showing an outer and inner dynein arm defect is shown in figure 3.5-1.

Figure 3.5-1 : EM of absent dynein arms

Fig 3.5-1: An electron micrograph picture showing the cross section of a cilia with absent outer and inner dynein arms.

It is important to be aware that secondary abnormalities of ciliary ultrastructure are common. These secondary abnormalities can be demonstrated in up to 10% of cilia from normal individuals (Afzelius 1981). Characteristic secondary

abnormalities are compound cilia and cilia with a variety of microtubule defects. The percentage of cilia with these abnormalities is increased in individuals who smoke and in those suffering from or recovering from a respiratory tract infection (Fox, Bull et al. 1983) (Buchdahl, Reiser et al. 1988). These secondary ciliary abnormalities have well documented affects on the ciliary beat frequency that can create diagnostic difficulties in PCD. Consequently each patient under investigation for PCD should have been free from respiratory infection for at least 4 - 6 weeks prior to performing ciliary studies.

Mucociliary clearance is assessed using either the saccharin test (Rutland and Cole 1981; Stanley, MacWilliam et al. 1984) or a radiolabelled assay (Quinlan, Salman et al. 1969). In both of these investigations the particle is placed on the

inferior turbinate of the nose 1cm from the anterior end and the patient is required to sit quietly with their head forward. They are asked to refrain from sniffing, coughing, sneezing, eating or drinking for the duration of the test. In the saccharin test they volunteer when they can taste the particle and the patients ability to taste saccharin is also confirmed by placing a particle of saccharin on the tongue after the investigation has been completed. A test lasting longer than 1 hour shows abnormal mucociliary clearance. This is a difficult investigation to perform accurately in young children but is a useful screening test for ciliary defects in older children and adults. In the case of radiolabelled albumin the progress of the particle is documented using a gamma camera and the duration of the test is much shorter. This test is widely used in Europe but is not popular in Great Britain.

To demonstrate ciliary ultrastructural and orientation abnormalities a ciliary brushing or biopsy is performed (Rutland, Griffin et al. 1981). A cooperative patient is first asked to blow their nose vigorously several times to clear the mucus secretions. The brushings can then be taken from the inferior or middle turbinate under direct vision using an oroscope in older children and adults. In younger children and infants the nose is cleared of secretions and the procedure is performed blind. Local anaesthetic can be used but anaesthetic agents have been demonstrated to influence the ciliary beat frequency (Gyi, O'Callaghan et al. 1994). The procedure is not painful but is uncomfortable and can be

distressing to younger children who may be unwilling to have it repeated. Biopsies are normally taken from the middle turbinate or the bronchus if fibreoptic bronchoscopy is being performed for additional reasons. A rare

complication is excessive bleeding from the site of biopsy especially in the case of the inferior turbinate. Patients should obviously be warned about this. A nasal ciliary brush is shown in Figure 3.5-2.

Figure 3.5-2: Nasal ciliary brush

1

Figure 3.5-2: A modified bronchiolar cytology brush used to take cilia lined epithelial cells from the turbinates of the nose.

The strips of epithelial cells obtained are placed in cell culture medium and then examined at a magnification of X 320 under light microscopy at 37 degrees. This should ideally occur within two hours of the brushing or biopsy being taken. The ciliary waveform and the character of the ciliary beat can be directly observed at microscopy and the ciliary beat frequency (CBF) measured. This can be done in a number of different ways. The first method to be described was a photometric technique in which the beating cilia periodically interrupt a narrow light source (Yager, Chen et al. 1978) (Rutland and Cole 1980). The fluctuation in light

intensity is then transduced into an electrical signal whose periodicity reflects the CBF. More recent techniques include use of a photodiode linked to computer software that allows the cilia to be visualised on screen. In most cases a normal ciliary beat pattern and CBF will exclude the diagnosis of PCD.

Further analysis by transmission electron microscopy to determine the

ultrastructural phenotype requires fixation of the sample in cacodylate-buffered 2.5% glutaraldehyde. The detailed fixation technique currently used at The Royal Brompton Hospital is described below in Table 3.5-3. A suggested algorithm for the investigation of patients suspected of having PCD is shown in Fig 3.5-3.

Table 3.5-3: Fixation technique for EM

1. Gluteraldehyde Fixative 50ml Sodium cacodylate 0.1 M

4ml HCL 0.1 M

2ml Calcium chloride 1% 10ml Gluteraldehyde 25% aqueous 2. This is made up to 100ml with distilled water

3. The samples are fixed in Gluteraldehyde

4. The samples are washed twice in cacodylate buffer

5. The buffer is replaced with osmium tetroxide and the samples are left for 1 hour 6. The osmium tetroxide is replaced with distilled water

7. The samples are centrifuged

8. The water is replaced with molten agar 9. The samples are spun and allowed to set

10. The samples are then dehydrated using methanol, proylene oxide and araldite

Figure 3.5-3: Algorithm for diagnosis of PCD

Child > 12yrs and Adult

Mucociliary clearance

I

I

I

i

I

i

Fig 3.5-3: A suggested algorithm for the diagnosis of PCD. CBF - ciliary beat frequency, EM electron microscopy

The view of the ciliary axoneme obtained at transmission electron microscopy is influenced by both the fixation technique (Fox, Bull et al. 1983) and the point at which cross sections are taken along each cilium (Chapter 1). The inner dynein arms are particularly hard to identify reliably and reproducibly. Interpretation of these results is therefore more reliable when performed in centres that have extensive experience in both the analysis and interpretation of normal and abnormal ciliary ultrastructure.

Orientation studies may be performed in cases where normal ultrastructure has been demonstrated. This involves determining the ciliary axis by comparing the alignment of the central microtubules as seen in transverse section on electron micrographs (De-longh and Rutland 1989). Ciliary disorientation (Rutland and De-longh 1990) (Rutman, Cullinan et al. 1992) has been postulated as an explanation for cases in whom a normal ultrastructure has been documented on electron microscopy but symptoms compatible with PCD and an abnormal mucociliary clearance or ciliary beat have also been demonstrated (Greenstone, Dewar et al. 1983). This diagnosis remains controversial as disorientation

accompanies some primary and secondary ciliary defects [(De-longh and Rutland 1989) but does also appear to occur as an isolated secondary

phenomenon (Rutman, Cullinan et al. 1992). An alternative explanation would be that subtle ultrastructural defects leading to disorientation may be missed with current EM technology. For example, a section of cilia prepared for EM analysis may be over 100 nm in depth. The basic repeat of the axoneme is 96nm. The classic cross sectional view is therefore composed of superimposed structures.

At present the diagnosis of PCD can be difficult. It is based on the exclusion of other differential diagnoses together with the demonstration of abnormal

mucociliary function occurring as a direct result of primary ciliary abnormalities. Newer methods to aid diagnosis continue to be evaluated. The level of nasal Nitric Oxide (NO) in the exhaled air from adults and children with PCD is markedly reduced and research is ongoing to establish whether NO will have a

future in screening individuals for PCD. NO is known to be an important

molecule involved in cell signalling. It is also known to be produced by activated macrophages and neutrophils to assist them in killing invading organisms which explains why it is increased in patients with asthma and bronchiectasis of

unknown cause. One explanation of its extremely low levels in patients with PCD may be that the ciliary immotility prevents it diffusing from the sinuses where it is produced into the upper respiratory tract. An alternative explanation would be that the production of NO is somehow dependent on normal ciliary function.

The establishment of in vitro cell culture systems in which epithelial cells are encouraged to regenerate cilia is another exciting development. These tissue culture systems have allowed confirmation of ciliary beat frequency and

ultrastructure without the effect of secondary changes due to in vivo infection. At present this technique remains a research tool but has implications for improving the availability of diagnostics.