Nitric oxide (NO) is a colorless, odorless gas produced in the upper and lower respiratory epithelium. It has diverse effects in the respiratory system, including vasodilation, bacterial killing, and the modulation of inflammation [12,13]. Fractional exhaled NO from the lower airways (FeNO) is normally upregulated in times of infection and inflammation (as often seen in poorly controlled asthma), while nasal NO is mainly produced in the paranasal sinuses.

Nasal nitric oxide (nNO) measurement represents a simple, non-invasive way to screen patients for PCD, with results immediately available at the time of testing. For unclear reasons, nNO values are distinctly reduced in individuals with PCD [14]. Other conditions that may have overlapping low nNO values include cystic fibrosis, diffuse pan-bronchiolitis, acute viral respiratory infection, and certain forms of primary immunodeficiency [15,16,17,18,19,20]. Thus, these conditions should always be considered and investigated if relying on low nNO as the sole diagnostic evidence for likely PCD (i.e., negative genetic testing and normal TEM analysis). When nNO values are repeated over time and remain persistently low, they can yield a provisional diagnosis of PCD, but cannot fully confirm the diagnosis and must be taken in context with other PCD diagnostic test results. It should be noted that only chemiluminescence NO devices have been prospectively validated for use in PCD investigations, with results from significantly less expensive electrochemical NO devices lacking robust studies in large PCD populations [21,22].

Cross-sectional and longitudinal observations have shown that ≥90% of individuals with PCD have nasal NO levels below 77 nL/min [18], when measured per a standardized protocol. Pooled sensitivity and specificity estimates for nNO < 77 nL/min are 96% and 96%, respectively, in cases of PCD that were confirmed using TEM and/or genetic analysis and when cystic fibrosis has been ruled out [23]. Low nasal NO values should be confirmed with at least one additional test on a separate visit, with patients at their baseline health, and without evidence of viral respiratory infection.

Around 5–10% of PCD cases have nNO values above the established cut-off of 77 nL/min, specifically those associated with mutations in radial spoke or central apparatus proteins [23,24]. These include cases due to genetic variants in RSPH1, which are associated with normal ciliary ultrastructure by TEM and nNO values of 100 to 300 nL/min [18] (Table 1).

Successful nNO testing requires a patient be cooperative enough to blow into a resistor device and achieve steady state NO measurement. Children under 5 years old often cannot perform this maneuver, so nNO testing may not be appropriate in preschool-aged children. Tidal breathing nNO measurements for patients 2–5 years old are feasible and are currently under investigation; however, diagnostic cut-off values are undefined in this age group. According to ATS guidelines, in patients with an appropriate PCD clinical phenotype and one nNO value <77 nL/min, a repeat measurement is recommended at least 2 weeks later [44]. With repeat low nNO values and negative cystic fibrosis testing, a presumptive diagnosis of PCD can be made and appropriate PCD therapies commenced. However, the availability of nNO testing is still hampered by the lack of clinical regulatory approval in North America; thus, most nNO tests are performed under research protocols in certified PCD centers, which are not reimbursed by insurance plans.

PCD is a genetically heterogenous disease caused by variants of more than 50 genes. This number will probably increase as more gene mutations are associated with PCD. The most frequently reported mutations occur in DNAI1 and DNAH5, which were the first implicated PCD genes [45,46]. The high number of pathogenic variants in different genes associated with PCD is a derivative of the complexity of the motile cilium, which is composed of hundreds of structural proteins and an unknown number of cytoplasmic factors that are required for ciliary assembly. Most PCD gene mutations are inherited in an autosomal recessive fashion. However, other inheritance modes occur, including x-linked inheritance in the assembly factor PIH1D3 and autosomal dominant inheritance in mutations of the forked head transcription factor FOXJ1 [33,47,48]. It is believed that mutations in known PCD-associated genes account for approximately 70–80% of PCD cases [49] (Figure 2).

Numerous commercial PCD genetic panels are available, varying in the number of genes covered and the employed sequencing technology. Most panels include the next generation sequencing analysis of at least 30 genes, with some offering more than 40 genes, plus deletion/duplication analysis. Panels covering greater numbers of PCD genes will have greater detection rates [50] (Figure 2). It is expected that the routine use of newer sequencing methods, such as whole exome sequencing (which offers better coverage), will likely replace panels with limited numbers of genes [51].

Although the increased availability of genetic tools is transformative for PCD diagnosis, interpreting the results of these tests may be challenging, due to the high number of variants of unknown significance (VUS), which often occur when there is discrepancy in the prediction tools used to determine pathogencity or when mutations were not previously reported to be pathogenic. PCD diagnosis requires two pathogenic variants be found in a single known PCD gene, on two opposite chromosomes (in trans) or on one allele, in the case of x-linked or autosomal dominant forms (PIH1D3, RPGR, OFD1, or FOXJ1). Pathogenicity of VUS results should be evaluated on a case-by-case basis, after consultation with either a geneticist or a PCD specialty center; VUS results cannot be assumed as disease-causing for diagnostic purposes.

Traditionally, the use of TEM to analyze ciliary ultrastructure was considered the gold standard for a diagnosis of PCD. Motile ciliary cross sections show a typical 9 + 2 arrangement (Figure 2) that represents 9 outer microtubule doublets surrounding a central pair. Outer dynein arms (ODA) and inner dynein arms (IDA) extend from the microtubule, acting as the motors powering ciliary movement. [52]. Radial spokes and nexin links provide stability to beating ciliary axonemes.

Procuring and processing TEM samples requires significant expertise. From sample collection considerations (i.e., nasal versus endobronchial scraping as opposed to “pinch” tracheal biopsies) to sample processing considerations (i.e., choice of fixatives and skill of technicians in choosing multiple ciliary tufts across the sample) the TEM image quality can vary greatly. Interpretation is similarly challenging and requires evaluation by pathologists with extensive experience examining ciliary ultrastructure. To address these challenges, a more recent international consensus guideline focused on reporting TEM results in the diagnosis of PCD, which included minimal criteria for acceptable TEM studies and the creation of various diagnostic classes of TEM defects. Per this protocol, only “hallmark” defects (class 1: absent ODA, absent ODA+IDA, and absent IDA with microtubular disorganization) are reliably diagnostic of PCD [53]. Ultimately, 30% of genetically proven cases of PCD do not have a hallmark defect on TEM [1], due to the limited sensitivity of EM in identifying small structural changes in the electron dense ciliary axoneme. This is especially true when identifying changes affecting the central pair, radial spokes, or inner dynein arms [54].

A promising diagnostic test using immunofluorescent-labeled antibody microscopy has been adopted in some European PCD centers [55]. Immunofluorescent antibody microscopy has the advantage of being more readily available than TEM, costing less per sample, and is possibly more sensitive than TEM depending on the panel of antibodies used. However, the diagnostic accuracy of immunofluorescent antibody testing has not been firmly established in PCD [55]. Most local pathology laboratories also possess the technical knowledge to process and interpret immunofluorescence antibody samples, as this technique is currently used in a variety of human diseases. Immunofluorescent antibody microscopy is currently not available for clinical use in North American centers, though this technique is being considered at several centers.