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. Author manuscript; available in PMC: 2025 May 1.
Published in final edited form as: Pediatr Pulmonol. 2024 Feb 21;59(5):1410–1417. doi: 10.1002/ppul.26929

The Utility of Nasal Nitric Oxide in the Diagnostic Evaluation of Primary Ciliary Dyskinesia

Katherine A Carr 1, Paul E Moore 1, Michael G O’Connor 1
PMCID: PMC11058016  NIHMSID: NIHMS1968070  PMID: 38380959

Abstract

There is no gold-standard test for primary ciliary dyskinesia (PCD), rather American Thoracic Society guidelines recommend starting with nasal nitric oxide (nNO) in children ≥5 years-old and confirming the diagnosis with genetic testing or ciliary biopsy with transmission electron microscopy (TEM). These guidelines have not been studied in a clinical setting. We present a case series describing the PCD diagnostic process at our pediatric PCD center. Diagnostic data from 131 patients undergoing PCD consultation were reviewed. In all participants ≥5 years-old and who completed nNO using resistor methodology, the first diagnostic test performed was nNO in 77% (73/95), genetic testing in 14% (13/95) and TEM in <1% (9/95). nNO was the only diagnostic test performed in 75% (55/73) of participants who completed nNO first. 75% (55/73) had a single above the cutoff nNO value and PCD was determined to be unlikely in 91% (50/55) without performing additional confirmatory testing. 11% (8/73) had multiple below the cutoff nNO values, with 38% (3/8) being diagnosed with PCD by confirmatory testing and 50% (4/8) with negative confirmatory testing, but being managed as PCD. The genetic testing positivity rate was 50% in participants who completed nNO first and 8% when genetic testing was completed first. nNO is useful in three situations: an initial above the cutoff nNO value makes PCD unlikely and prevents additional confirmatory testing, repetitively below the cutoff nNO values without positive confirmatory testing suggests a probable PCD diagnosis and the yield of genetic testing is higher when nNO is performed first.

Keywords: diagnostic algorithm, guidelines, initial above the cutoff nasal nitric oxide, repetitively below the cutoff nasal nitric oxide, screening

Introduction

Primary ciliary dyskinesia (PCD) is a rare, inherited disorder of motile cilia characterized by recurrent oto-sino-pulmonary infections beginning in early childhood. Despite the early onset of clinical symptoms, the average age of diagnosis is about 6 years of age1 and it is estimated that only 10% of people with PCD have been formally diagnosed2. Clinical signs and symptoms of PCD include unexplained neonatal respiratory distress, daily nasal congestion, daily cough, recurrent otitis media and organ laterality defects3-6. These symptoms can also be seen in more common pulmonary diseases; thus diagnostic testing is needed to confirm PCD. At present in North America, diagnostic testing options include: genetic evaluation, ciliary biopsy with transmission electron microscopy (TEM) and nasal nitric oxide testing (nNO).

There is no gold standard diagnostic test for PCD as each modality lacks sufficient diagnostic sensitivity and specificity7. PCD is associated with extensive locus and allelic genetic heterogeneity, which contributes to difficulties with genetic testing. There are currently 50 described disease-causing genes which are estimated to account for only 70% of patients with PCD, as more genes have yet to be discovered8,9. Additionally, few commercial genetic panels test for all known PCD genes10. TEM evaluates for disease causing ultrastructural defects within individual cilia, which has several limitations related to challenges with processing and interpretation. Samples need to be collected when patients are at their baseline as inflammation associated with acute infection or other insults can induce secondary changes in ciliary ultrastructure that appear similar to PCD11. It is estimated that 30% of patients with PCD have normal or non-diagnostic ciliary ultrastructure on EM12. When nNO is performed with a standardized protocol in the appropriate clinical context, values ≤77 nL/min are 96.3% sensitive and 96.4% specific for PCD13. Thus, nNO is a helpful first-line test to raise or lower the suspicion for disease. Despite being non-invasive and accurate, nNO testing requires specialized equipment, trained personnel and standardized training protocols which can make access to accurate testing difficult14. nNO can also be low in conditions outside of PCD including immunodeficiency, cystic fibrosis and acute viral respiratory infections15. Additionally, the American Thoracic Society does not formally recommend using nNO in children under 5 years of age, but the European Respiratory Society recently published technical standards for nNO that recognized its utility as a diagnostic testing modality in this age group using a lower cutoff value of 44 nL/min in children 2-5 years of age16.

In 2018, the ATS published a diagnostic algorithm for evaluating suspected PCD in North America that recommends using a panel of diagnostic tests7. This algorithm also takes into consideration the age of the participant and the availability of testing modalities. In this guideline, diagnosing PCD starts with recognizing the clinical phenotype, followed by nNO testing when available and appropriate then confirming the diagnosis with an extended genetic testing panel and lastly TEM if indicated. Of note, nNO is only recommended as a first line test when cystic fibrosis has been ruled out, in cooperative children ≥ 5 years of age and when performed at a specialty center using a chemiluminescence device via a standardized protocol with resistor techniques. These guidelines have not yet been studied in a real-world, clinical setting. We present in this manuscript a comprehensive case series describing our pediatric PCD Foundation accredited center’s experience in incorporating nNO into the diagnostic evaluation of PCD.

Methods

At the time of PCD Foundation center accreditation in 2017, we established nNO testing and a prospective patient registry. All patients who were referred to our center for considerations of PCD were enrolled in this prospective registry at time of consultation (IRB # 160951; Vanderbilt University Medical Center). All participants signed written informed consent or assent when applicable for both completion of nNO testing as well as collection of prior PCD diagnostic testing and relevant clinical symptoms. Specific information collected included demographics, PCD diagnostic status, alternative non-PCD diagnoses, nNO results, TEM results, genetic testing results and medication and treatment strategies utilized. Of note, as this is a prospective observation of patients referred to our PCD center, some patients had PCD diagnostic testing prior to referral and some had no PCD targeted testing. A retrospective analysis of registry data from all people enrolled between 2018 and 2022 was performed. Per PCDF standard operating procedure, nNO testing was completed using resistor or tidal breathing techniques13. A nNO cutoff value of 77 nL/min was used when interpreting results from resistor techniques and children ≥ 5 years of age14. Results obtained from tidal breathing techniques or children < 5 years of age were evaluated on a case-by-case basis as there is no standardized cutoff value for these groups. PCD diagnostic status was determined based on provider interpretation of clinical phenotype and diagnostic testing results. Confirmatory diagnostic testing was defined as PCD targeted genetic testing and/or TEM.

Results

Since PCD Foundation center accreditation in 2017, 131 patients have been referred to our center for PCD evaluation. The age at initial consultation ranged from 1 to 65 years with a median age of 8 years. Sex was evenly distributed and the vast majority of patients were white and not Hispanic/Latino (Table 1). Ultimately PCD was determined to be unlikely in 74% (97/131) of participants, 15% (19/131) are completing ongoing diagnostic evaluation for PCD and 11% (14/131) were given a confirmed PCD diagnosis.

Table 1:

Demographics

Median Age at Presentation in Years 8
Sex 67 Males (51%)
63 Females (48%)
1 Unknown (1%)
Ethnicity 123 Not Hispanic/Latino (94%)
4 Hispanic/Latino (3%)
4 Unknown (3%)
Race 116 White (89%)
7 Black (5%)
3 Other (2%)
2 Asian (2%)
2 Unknown (2%)
1 Native Hawaiian/Pacific Islander (1%)
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nNO testing was performed in 95% (124/131) of PCD consultations. The median age of participants who completed nNO was 9 years and 83% (103/124) were ≥5 years of age. Regarding methodology, 77% (96/124) used resistor techniques and 23% (28/124) tidal breathing. The median initial nNO result was 217 nL/min in all participants who completed nNO, 237 nL/min in participants who utilized resistor techniques and were >5 years old and 126 nL/min in participants who utilized tidal breathing techniques and/or were <5 years of age.

Among our study population, 73% (95/131) of patients were ≥5 and if nNO was performed it was completed using resistor techniques. nNO testing was the first test performed in 77% (73/95) (Figure 1), genetic testing was performed first in 14% (13/95) (Figure 2) and TEM was performed first in <1% (9/95) (Figure 3).

Figure 1:

Figure 1:

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Diagnostic Process in Participants who Completed nNO Testing as the Initial Diagnostic Test

Figure 2:

Figure 2:

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Diagnostic Process in Participants who Completed Genetic Testing as the Initial Diagnostic Test

Figure 3:

Figure 3:

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Diagnostic Process in Participants who Completed Ciliary Biopsy with Transmission Electron Microscopy as the Initial Diagnostic Test

nNO as a First Diagnostic Test

nNO was the only diagnostic test performed in 75% (55/73) of participants who completed nNO first. Ultimately 12% (9/73) were diagnosed with PCD based on confirmatory testing, PCD was determined to be unlikely in 81% (59/73) and 7% (5/73) are completing ongoing diagnostic evaluation. 75% (55/73) of participants had a single above the cutoff nNO value. PCD was determined to be unlikely in 91% (50/55) of this group without performing any additional PCD targeted testing and 94% (47/50) were given an alternative, non-PCD diagnosis based on the results of their initial consultation. Primary alternative diagnoses were 55% asthma, 17% immune dysfunction, 11% non-allergic rhinitis, 4% interstitial lung disease, 4% aspiration, 4% eosinophilic esophagitis, 2% allergic rhinitis and 2% allergic bronchopulmonary aspergillosis. 11% (8/73) of participants had multiple below the cutoff nNO levels and 38% of this group (3/8) were diagnosed with PCD based on confirmatory diagnostic testing. 50% (4/8) of patients with multiple below the cutoff nNO levels had negative confirmatory testing. Of note, all of these patients are being clinically managed like they have PCD with antibiotics for respiratory exacerbations and routine airway clearance therapies. The genetic testing positivity rate in participants who completed nNO as the first diagnostic test was 50% (9/18) (Table 2). The TEM positivity rate was 0% (0/2) when nNO was used as an initial diagnostic test.

Table 2:

Confirmatory Testing Positivity Rate

Genetic Testing Positivity Rate When Initial Diagnostic Test is nNO 50%
Genetic Testing Positivity Rate When Initial Diagnostic Test is Genetic Testing 8%
Genetic Testing Positivity Rate When Initial Diagnostic Test is TEM 0%
TEM Positivity Rate When Initial Diagnostic Test is nNO 0%
TEM Positivity Rate When Initial Diagnostic Test is Genetic Testing 33%
TEM Positivity Rate When Initial Diagnostic Test is TEM 8%
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Genetic Testing First Diagnostic Test

Genetic testing was the only test performed in 8% (1/13) of participants who completed genetic testing first. 85% (11/13) of participants completed nNO testing in addition to genetic testing. Ultimately 8% (1/13) were diagnosed with PCD, PCD was determined to be unlikely in 70% (9/13) and 23% (3/13) are completing ongoing diagnostic evaluation. The genetic testing positivity rate when genetic testing was used as an initial diagnostic test was 8% (1/13). The TEM positivity rate when genetic testing was performed first was 33% (1/3).

Ciliary Biopsy with Transmission Electron Microscopy First Diagnostic Test

All participants who completed TEM as their first diagnostic test required additional testing modalities in order to achieve a PCD diagnostic decision. Of note, nNO was also utilized in all patients who completed TEM as a first test. 56% (5/9) of participants had inconclusive TEM results. Ultimately 11% (1/9) were diagnosed with PCD and PCD was determined to be unlikely in 89% (8/9). The genetic testing positivity rate when TEM was performed first was 0% (0/3). The TEM positivity rate when TEM was used as an initial diagnostic test was 8% (1/11).

Participants Under 5 Years Old and/or Completed nNO Using Tidal Breathing

27% (36/131) of our study population consisted of children <5 years of age and/or individuals who completed nNO using tidal breathing methodology (Figure 4). 83% (30/36) of this population completed nNO and all children were between ≥2 years of age. 25% (9/36) of this population was ≥5 years of age, but was unable to perform nNO using resistor methodology as participants were either unable or unwilling to generate a reproducible plateau for >3 seconds. nNO was the first diagnostic test performed in 39% (14/36), genetic testing was the first diagnostic test performed in 39% (14/36) and TEM was the first test performed in 22% (8/36). Among all patients who completed nNO, 27% (8/30) had an initial value ≤44 nL/min, 10% (3/30) had an initial value 44-77 nL/min and 63% (19/30) had an initial value ≥77 nL/min. Among the group with an initial nNO value <44 nL/min 13% (1/8) were diagnosed with PCD, PCD was determined to be unlikely in 50% (4/8) and 38% (3/8) are completing ongoing PCD evaluation. Among the group with an initial nNO value 44-77 nL/min 0% were diagnosed with PCD, PCD was determined to be unlikely in 66% (2/3) and 33% (1/3) are completing ongoing PCD evaluation. Among the group with an initial nNO value >77 nL/min 0% were diagnosed with PCD, PCD was determined to be unlikely in 90% (7/19) and 10% (2/19) are completing ongoing PCD evaluation. Of note, 38% (3/8) of the group with an initial nNO value ≤44 nL/min and 33% (1/3) of the group with an initial nNO value 44-77 nL/min had normalizing values over time.

Figure 4:

Figure 4:

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nNO Values in Children <5 years old and/or Who Completed the Procedure Using Tidal Breathing Methodology

Discussion

Making a diagnosis of PCD is challenging in part due to the absence of a gold-standard diagnostic test, but nNO testing has an important role in the diagnostic process. In this manuscript, we describe our PCD Foundation accredited center’s real-world experience with nasal nitric oxide (nNO) testing. At our center, nNO has been routinely implemented into the diagnostic evaluation of PCD as an initial test for all patients who have clinical signs/symptoms consistent with PCD. In our clinical experience, nNO seems to have the highest diagnostic utility in three scenarios: 1) nNO as an initial diagnostic step for PCD evaluation improves our yield of confirmatory testing such as genetic analysis, 2) nNO as first line test quickly helps lower the suspicion for PCD allowing providers to make alternative diagnoses, 3) in individuals with a PCD clinical phenotype, but non-confirmatory genetic testing or TEM results, repetitively below the cutoff nNO levels allows us to make a clinical PCD diagnosis and start disease specific therapies.

Despite a robustly described PCD clinical phenotype, the symptoms of PCD overlap with other common diseases17. Thus, initial nNO results in the appropriate clinical context can help stratify the likelihood of a PCD diagnosis. PCD diagnostic guidelines show that when nNO results are above the cut-off value of 77 nL/min, a diagnosis of PCD is much less likely7. Our data confirms this as most patients who completed nNO testing only once with a value >77 nL/min were given an alternative diagnosis to explain their chronic respiratory symptoms. Importantly, the use of nNO testing was helpful in quickly reaching an alternative specific disease diagnosis.

Although a single, above the cutoff nNO value can be useful in lowering the suspicion for PCD in most patients, it is important to interpret these results on a case-by-case basis. The current literature describes several PCD genotypes that are associated with normal nNO levels18. There was 1 patient in our cohort with a nNO value of 79.0 nL/min who was diagnosed with PCD based on confirmatory testing, which further supports this point. This patient had pathogenic variants in CCNO, which has been reported to have normal nNO levels19,20. This scenario highlights the notion supported in the 2018 ATS guidelines that when there is a strong suspicion for PCD, normal nNO testing should not prevent further PCD diagnostic testing.

Incorporating nNO into PCD evaluation as an initial test improved our genetic testing positivity rate. When nNO was performed as an initial diagnostic test in in children ≥5 years old using resistor methodology, the genetic testing positivity rate was substantially higher (50%) than when genetic testing was performed as an initial diagnostic test (8%) or when genetic testing was performed after TEM (0%). This shows that in the correct clinical context, a below the cutoff nNO result can improve the PCD detection rate for genetic testing. As nNO has great diagnostic accuracy for PCD with a sensitivity of 96.3% and specificity of 96.4%,13 one might expect that the genetic testing positivity rate in our group of patients with below the cutoff nNO levels to be much higher. This discrepancy is likely explained by issues with generalizability within the current nNO literature and limitations inherent to genetic testing. The current sensitivity and specificity values for nNO are based on a highly curated population who had a high likelihood of PCD based on clinical features and used a main reference standard of TEM ultrastructural defects. As we know that TEM identifies more classic forms of PCD with below the cutoff nNO levels, we may be missing variant forms in these analyses.13 Thus, the results of these studies are less generalizable and sensitivity and specificity values may be lower than reported when applied to a real world, clinical setting. Current commercially available genetic testing panels test for 39-47 PCD associated genes,10 and even if genetic panels included all known causative genes, these 50 genes are only thought to detect 70% of patients with PCD7.

Although the ATS guidelines only recommend using nNO in children ≥5 years of age using resistor mechanisms, evidence suggests that nNO still has utility in younger age groups, but a lower cut-off value may need to be considered. All infants have very low nNO levels at the beginning of life, but healthy infants have increasing nNO values over time.21 At 2 years of age, there is a statistical difference in nNO values in children with PCD compared to healthy controls, but the difference between these groups is smaller compared to older children and adults.22 Additionally, a European Respiratory Society Task Force recently published updated technical standards for nNO testing in PCD which recommended performing the procedure in all age groups, but suggested a cut-off value of 30 nL/min in children 0-2 years of age and a cutoff of 44 nL/min in children 2-4 years of age.16 It is important to note that these recommendations were based mostly on experience rather than extensive multicenter evidence. The results from our patient population <5 and/or who completed nNO via tidal breathing support the idea that nNO values are inherently lower. The median initial nNO level in this population was substantially lower than participants who completed resistor techniques. Additionally, 36% (4/11) of participants with initial nNO values <77 nL/min had normalizing values over time, highlighting that results in this population must be interpreted with extreme caution. Our results also support the idea that interpreting nNO using a lower cutoff value of 44 nL/min may have diagnostic utility, as the only patient who was ultimately diagnosed with PCD in this group had an initial nNO level of 28 nL/min. This also highlights the need for additional research in establishing age specific nNO cutoff values in this population. Despite these pitfalls, it is important to note than an initial above the cutoff nNO value in this population would be reassuring against PCD.

Trending nNO levels over time in patients with clinical features of PCD also aids in diagnosis and management. Both genetic testing and TEM detects only about 70% of patients with PCD. In patients with a PCD phenotype, but without positive confirmatory diagnostic testing, repetitively below the cutoff nNO levels allow physicians to make a probable PCD diagnosis and start PCD targeted therapies. At our institution, all patients with repetitively below the cutoff nNO levels are being managed similarly to patients with PCD with daily airway clearance and IV and inhaled antibiotics during respiratory exacerbations. Additionally, nNO levels can normalize overtime which is helpful in suggesting that PCD is unlikely. nNO levels can be transiently below the cutoff during acute viral respiratory infections, then normalize after infection resolution15. Patients with immunodeficiencies can also have intermittently below the cutoff nNO levels23. nNO values may also normalize overtime due to improvement in testing technique and with age, as discussed previously.

There are some additional important comments to make based on our data. The majority of consultations did not result in a PCD diagnosis, which is common in diagnosing rare diseases. Even though PCD is a racially diverse disease with individuals of African descent having the highest prevalence of disease-causing variants24, most patients referred to our center for PCD evaluation were white (89%) and non-Hispanic/Latino (94%). The lack of diversity in our PCD consultation population is a limitation to the generalizability of our study, but highlights the problem of racial inequity in the diagnosis of PCD. Targeted and impactful efforts are required to further understand and combat this problem in the North American PCD population. When analyzing the diagnostic process for all patients in our study, on numerous occasions the ATS 2018 guidelines were not precisely followed, which is a limitation. Although the current guidelines recommend confirming a below the cutoff nNO level by repeating the procedure, several of our participants underwent confirmatory diagnostic testing instead. Additional algorithm deviations included performing nNO in children under 5 years of age, using TEM as a first line confirmatory test instead of genetic testing and performing confirmatory testing as a first line test in some children over 5 years of age. Although the reasons for these algorithm derivations were not thoroughly explored, some of them can be explained by the fact that initial PCD diagnostic testing was performed at other institutions (some without access to nNO) prior to referral to our PCD center where nNO is available. Regardless of the etiology, the presence of these deviations emphasize the need for the continued evaluation of these guidelines in the real world setting in order to understand how factors such as barriers to follow up and high initial clinical suspicion for PCD may influence providers to make alternative diagnostic decisions.

This manuscript describes our pediatric PCD Foundation accredited center’s real-world experience with nNO testing, specifically highlighting how nNO is utilized, challenges in interpretation and advantages in incorporating nNO into clinical practice. Based on our center’s experience, we have found nNO to be the most useful in 3 scenarios: 1) In the correct clinical context, an initial above the cutoff nNO level can quickly lower the suspicion for PCD, preventing costly, unnecessary confirmatory diagnostic testing and allowing for timely identification of non-PCD diagnoses, 2) in patients with a PCD clinical phenotype but with non-confirmatory genetic testing or TEM, repetitive below the cutoff nNO levels allows for establishing a probable PCD diagnosis and initiating PCD targeted therapies and 3) using nNO as an initial diagnostic step improves the genetic testing positivity rate.

Acknowledgments

The authors have indicated no potential conflicts of interest to disclose. KAC was support by a grant from the National Institutes of Health (NIGMS NIH T32GM007569, PI Bjorn Knollman, MD, PhD). All patients who were referred to our center for considerations of PCD were enrolled in a prospective registry at the time of consultation (IRB #160951; Vanderbilt University Medical Center). All participants signed written informed consent or assent when applicable for both completion of nNO testing as well as collection of prior PCD diagnostic testing and relevant clinical symptoms. This work was presented as a poster at the American Thoracic Society 2023 International Conference on May 23, 2023 in Washington D.C. KAC takes responsibility for the integrity of the data and accuracy of data analysis. KAC contributed substantially to the study design, data analysis and interpretation and writing of the manuscript. PEM and MGO contributed substantially to the study design, data interpretation and critical review of the manuscript.

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