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Introduction Primary ciliary dyskinesia (PCD) is a rare disease (∼1 in 15 000 people) inherited in a genetically heterogeneous, autosomal recessive pattern [1–3]. It is characterised by chronic infection of the upper and lower airways caused by impaired mucociliary clearance as a consequence of abnormal function of motile cilia. In healthy individuals, cilia clear airway mucus, bacteria and debris by coordinated beating. The ciliary dysfunction in PCD leads to a daily wet cough, recurrent chest infections and rhino-sinusitis. By adulthood, bronchiectasis is invariable and many patients develop respiratory failure [4]. Motile cilia are important in organ systems besides the airways, such as the embryonic node, sperm flagella and the female reproductive tract. Therefore, patients frequently have problems caused by non-respiratory dysmotile cilia, e.g. serous otitis media (“glue ear”) leading to hearing impairment and immotile sperm causing infertility). The cilia of the embryonic node, responsible for left–right asymmetry of organs, are similar in structure to respiratory cilia. Dysfunction of embryonic nodal cilia in PCD causes laterality defects, including situs inversus (chest and abdominal organs are mirror image of normal; seen in approximately 40% of cases) and situs ambiguous (disturbance of the usual left and right distribution of the thoracic and abdominal organs which does not entirely correspond to mirror image; seen in approximately 10% of cases) and can be associated with congenital heterotaxic heart disease in approximately 2–3% of cases [5].

imately 40% of cases) and situs ambiguous (disturbance of the usual left and right distribution of the thoracic and abdominal organs which does not entirely correspond to mirror image; seen in approximately 10% of cases) and can be associated with congenital heterotaxic heart disease in approximately 2–3% of cases [5]. Monitoring of disease progression and evaluation of therapeutic options has been hampered by a lack of disease-specific outcome measures. Spirometry is an insensitive marker of progressive lung disease, which is evident using high-resolution computed tomography (HRCT) [6]. Although HRCT is a useful staging test, it is an impractical monitoring tool. Lung clearance index (LCI), measured by multiple breath washout, has been investigated as a potential tool for monitoring at specialist centres using sulphur hexafluoride (SF6) as a tracer gas [7, 8]. However, contrary to findings in cystic fibrosis (CF), LCI does not appear to be a sensitive test of airway disease in advanced PCD [7]. Furthermore, physiological measures provide information on objective indicators of health to patients and clinicians, but these measures do not reflect the patient perceptions of the impact of the disease on symptoms as well as physical, social and emotional functioning.

tive test of airway disease in advanced PCD [7]. Furthermore, physiological measures provide information on objective indicators of health to patients and clinicians, but these measures do not reflect the patient perceptions of the impact of the disease on symptoms as well as physical, social and emotional functioning. Thus, measures are needed to assess the impact of PCD, from the patient's perspective, on all domains of patient functioning [9–11]. Health-related quality of life (HRQoL) measures have become a vital and necessary component of patient-reported outcomes (PROs) in populations with chronic disease [12]. The US Food and Drug Administration (FDA) defines HRQoL as the patient's perception of how they “survive, feel, and function” [13]. We used a model for HRQoL originally proposed by Wilson and Cleary [14] and revised by Ferrans et al. [15]. There is extensive agreement that assessment of HRQoL should encompass, at minimum, physical, social and emotional well-being and symptoms which allow for a multidimensional, systematic measure of how the illness and its treatment impact symptoms and other domains of functioning.

14] and revised by Ferrans et al. [15]. There is extensive agreement that assessment of HRQoL should encompass, at minimum, physical, social and emotional well-being and symptoms which allow for a multidimensional, systematic measure of how the illness and its treatment impact symptoms and other domains of functioning. Existing PROs do not assess the disease-specific effects of PCD on daily symptoms and functioning. Most studies have utilised either generic (e.g. Short Form 36 Health Survey) or broad-based respiratory questionnaires, such as the St George's Respiratory Questionnaire (SGRQ) [16–19] and two additional studies have utilised qualitative interview methods [20, 21]. These PROs have a number of limitations for assessing HRQoL in adults with PCD. For example, the SGRQ was developed for patients with chronic obstructive airways disease and, thus, has a limited number of respiratory symptoms, no items relevant to ear, nose and throat disease or fertility problems, long and variable recall periods and considerable respondent burden (e.g. nearly an hour to complete). Thus, there was an urgent need to develop a disease-specific measure for adults with PCD. These tools can be used to document the progression of disease, monitor patients clinically and serve as an outcome measure for clinical trials of new therapies.

nd considerable respondent burden (e.g. nearly an hour to complete). Thus, there was an urgent need to develop a disease-specific measure for adults with PCD. These tools can be used to document the progression of disease, monitor patients clinically and serve as an outcome measure for clinical trials of new therapies. Our ultimate goal is to develop a PCD-specific HRQoL instrument for use as a primary or secondary outcome measure in large, randomised clinical trials. To recruit adequate numbers of participants, multi-centre and multi-national collaboration was required. Therefore, researchers from the UK, North America and Ireland worked closely to develop age-specific questionnaires (child, adolescent, parent-proxy and adult) using guidance developed by the FDA and European Agency for the Evaluation of Medicinal Products (EMEA) [13, 22, 23]. Development of the child, adolescent and parent-proxy versions will be reported in a separate manuscript. This manuscript describes the development process for the QOL–PCD Adult version which included the following phases: 1) literature review and expert panels; 2) open-ended interviews with patients in in UK, USA and Canada; 3) item generation; 4) cognitive testing; and 5) refinement of the draft measure. QOL–PCD is now being validated in Europe, USA and Canada; the psychometric reliability and validity will be reported when these studies are complete.

view and expert panels; 2) open-ended interviews with patients in in UK, USA and Canada; 3) item generation; 4) cognitive testing; and 5) refinement of the draft measure. QOL–PCD is now being validated in Europe, USA and Canada; the psychometric reliability and validity will be reported when these studies are complete. Methods The protocol for development of the QOL–PCD complied with the FDA and EMEA requirements. The study was approved by Southampton and South West Hampshire Research Ethics Committee (Southampton, UK) A 07/Q1702/109 and by the University of Miami Institutional Review Board (Miami, FL, USA). Informed consent was obtained prior to interviews. Literature review and expert panel First, a systematic literature review was conducted to identify key symptoms and effects of PCD on patient functioning. MEDLINE and EMBASE were searched, and additional references were sought through citations in the identified studies. Abstracts were reviewed and manuscripts sourced for research investigating the effects of PCD on adults. In the next step, expert clinicians, allied health professionals and researchers met to discuss their own perceptions of the impact of PCD on adults, based on their clinical experiences. These sources of information contributed to a long list of items patients rated for relevance. These items also informed the development of the open-ended interview guide.

llied health professionals and researchers met to discuss their own perceptions of the impact of PCD on adults, based on their clinical experiences. These sources of information contributed to a long list of items patients rated for relevance. These items also informed the development of the open-ended interview guide. Participants In the UK, participants for the open-ended and cognitive interviews were recruited from PCD clinics and from an advert through the PCD Family Support Group UK. A list of potential items was sent to the Family Support Group in the UK to rate their relevance. Participants in North America were recruited from a cohort of PCD patients evaluated at the University of North Carolina, as well as from the US PCD Foundation's registry of patients. Expert opinion was also sought during the item generation and item reduction phases from members of the European PCD Group. Criteria for participation in the open-ended and cognitive interviews included age ≥18 years with a diagnosis of PCD. Patients were recruited from English-speaking countries: UK, USA and Canada. UK participants had an existing diagnosis from one of the English diagnostic centres [1, 24] based on clinical phenotype plus high-speed video analysis of ciliary function and/or assessment of ciliary ultrastructure by electron microscopy. North American participants were diagnosed at a specialised PCD research center, based on: a compatible clinical phenotype plus defect in ciliary ultrastructure and/or identification of biallellic disease-causing mutations in one of the PCD genes.

tion and/or assessment of ciliary ultrastructure by electron microscopy. North American participants were diagnosed at a specialised PCD research center, based on: a compatible clinical phenotype plus defect in ciliary ultrastructure and/or identification of biallellic disease-causing mutations in one of the PCD genes. Open-ended interviews In-depth interviews were conducted either in-person or by phone to elicit the effects of PCD from the patients' perspective. Interviews were conducted in the UK by L. Behan and in USA and Canada by A.L. Quittner, A. Alpern and A.M. Morris. All participants were fluent in English. We attempted to interview patients who were geographically representative and clinically stable. The audio of all interviews was recorded and transcribed for content analysis using either NVivo (version 8 2008; QSR International Pty Ltd, Daresbury, UK) in the UK or Atlas.ti (Version 7.0; Scientific Software Development, Corvallis, OR, USA) in the USA. Thematic coding was used to identify key symptoms and psychosocial impacts. Two members of each research team coded the interview transcripts using consensus coding. When there was a discrepancy, this was discussed and resolved within the pair of coders. We did not calculate percentage agreement since we used a consensus coding process. These data were then analysed to identify critical items based on frequency of endorsement. Content analysis of these transcripts yielded saturation and indicated that the measure was comprehensive and that all relevant items were included.

id not calculate percentage agreement since we used a consensus coding process. These data were then analysed to identify critical items based on frequency of endorsement. Content analysis of these transcripts yielded saturation and indicated that the measure was comprehensive and that all relevant items were included. Item generation Questions from the literature review, expert panel and open-ended interviews were also sent by post to respondents of an advert circulated to members of the PCD Support Group and to adult patients at PCD clinics in the UK. A pre-paid envelope and covering letter was included. Participants rated each item using a 5-point Likert scale (1: “not relevant”; to 5: “highly relevant”).

ended interviews were also sent by post to respondents of an advert circulated to members of the PCD Support Group and to adult patients at PCD clinics in the UK. A pre-paid envelope and covering letter was included. Participants rated each item using a 5-point Likert scale (1: “not relevant”; to 5: “highly relevant”). Items for the initial QOL–PCD measure were based primarily on the patient-based content analysis. We discussed these results in a series of teleconferences chaired by J.S. Lucas. Each meeting included clinicians, psychologists and interviewers from Ireland, the UK and the USA. Decisions made using a modified Delphi approach guided by two main principles. The primary criterion for including an item was its impact on HRQoL, measured by the frequency with which items were mentioned across patients and in relation to other items mentioned. There was no pre-determined frequency required for inclusion of an item, but the researchers considered the frequency each item was mentioned relative to the other items in that domain. They also considered the importance interviewees placed on these items. Secondly, patients' ratings of relevance from the UK survey were considered. For each item, decisions were made to include an item based on our discussion. When there was initial disagreement, the chair invited each individual to explain their rationale. Unanimous agreement was achieved within two rounds of discussion. Finally, we determined that all of the content had been identified based on saturation matrices in the UK and US, which showed that no new content emerged after six patients, on average, in each country. We included at least four items on each subscale to ensure adequate internal consistency.

two rounds of discussion. Finally, we determined that all of the content had been identified based on saturation matrices in the UK and US, which showed that no new content emerged after six patients, on average, in each country. We included at least four items on each subscale to ensure adequate internal consistency. Construct of prototype questionnaire Agreement on item selection and wording was achieved during multi-disciplinary, multinational conference calls. Selected items were written using patient language obtained in the qualitative interviews and then combined into scales (e.g. frequency and severity of respiratory symptoms, perceptions of treatment burden). Where appropriate, the items were formulated into questions based on the Cystic Fibrosis Questionnaire-Revised (CFQ-R), which has been well-validated [12, 25]. Some example CFQ-R items that were adapted to the PCD HRQoL measure include: “Did you cough during the day?” and “How often does CF get in the way of meeting your school, work, or personal goals?” Items were written to ensure conceptual, cultural and linguistic equivalence for North America, the UK and Ireland, by researchers from the USA, England and Ireland. We also adhered to both the FDA and EMEA Guidance [13, 22, 23]. For example, as recommended, we asked patients to answer questions based on their symptoms and impact over the past week.

nceptual, cultural and linguistic equivalence for North America, the UK and Ireland, by researchers from the USA, England and Ireland. We also adhered to both the FDA and EMEA Guidance [13, 22, 23]. For example, as recommended, we asked patients to answer questions based on their symptoms and impact over the past week. Cognitive interviews Cognitive interviews were conducted prior to formal psychometric validation of questionnaires to evaluate how respondents process the question and rating options cognitively; i.e. What “meaning” do these items have for respondents? Is this the same meaning we intended? What were they considering when rating frequency or impact? Specifically, we wanted to identify any problems with the instructions, organisation of the questionnaire, item interpretation, memory retrieval, decision-making processes and response selection. A “think aloud” procedure was used to investigate the participants' comprehension of the instructions, items and rating scales. They were asked clarifying questions, such as: “What were you thinking of when answering that question?” and “What does X word mean to you?” For example, “What does feeling ‘well’ mean to you?”, “What would have made you endorse a higher/lower frequency for that question?”, “How relevant/important is this question for you?”, “Are the rating options clear to you?” and “Are they easy to use?”

when answering that question?” and “What does X word mean to you?” For example, “What does feeling ‘well’ mean to you?”, “What would have made you endorse a higher/lower frequency for that question?”, “How relevant/important is this question for you?”, “Are the rating options clear to you?” and “Are they easy to use?” Participants were first asked to complete the prototype questionnaire independently. Next, they were interviewed using specific cognitive probes, which focused on the clarity of the question, its meaning, relevance and importance, and what would have shifted their response to an adjacent answer. The audio of all interviews was recorded and transcribed. The results were discussed during a series of teleconferences to determine whether revisions were required for the format, instructions or items. The measure was refined based on the cognitive interviews and then finalised.

their response to an adjacent answer. The audio of all interviews was recorded and transcribed. The results were discussed during a series of teleconferences to determine whether revisions were required for the format, instructions or items. The measure was refined based on the cognitive interviews and then finalised. Results Item generation Items were generated by the expert panel and the qualitative, open-ended interviews with patients. Characteristics of participants who completed the open-ended interviews (n=21) are shown in table 1. The majority of participants were female, and among US participants, most were between 18 and 35 years of age. As expected, nearly all adults described a chronic cough and sino-nasal symptoms. Eight (38%) participants described themselves as infertile or had required assisted fertilisation, but 10 (48%) participants had not yet tried to conceive or had their fertility status checked. Selected patient quotes from the open-ended interviews conducted with UK and North American participants are presented in table 2. TABLE 1 Demographics and clinical characteristics of participants

ired assisted fertilisation, but 10 (48%) participants had not yet tried to conceive or had their fertility status checked. Selected patient quotes from the open-ended interviews conducted with UK and North American participants are presented in table 2. TABLE 1 Demographics and clinical characteristics of participants Study population UK USA Participants n 21 11 10 Sex Male 3 (14) 1 (9) 2 (20) Female 18 (86) 10 (91) 8 (80) Age years 18–35 12 (57) 5 (45) 7 (70) 36–50 4 (19) 1 (9) 3 (30) 51–65 3 (14) 3 (27) >65 2 (10) 2 (18) Age at diagnosis years <5 4 (19) 1 (9) 3 (30) 5–12 4 (19) 0 4 (40) 12–18 1 (5) 1 (9) 0 >18 12 (57) 9 (82) 3 (30) Race/ethnicity UK White British 9 (82) British Asian 1 (9) Asian 1 (9) USA Caucasian 10 (100) Hispanic 9 (90) White non-Hispanic 1 (10) Symptoms Chronic wet cough 21 (100) 11 (100) 10 (100) Persistent runny nose 20 (95) 11 (100) 9 (90) Recurrent sinus disease 16 (76) 8 (72) 8 (80) Infertility 8 (38) 4 (36) 4 (40) Situs abnormalities 12 (57) 8 (72) 4 (40) Cardiac disease 0 0 0 FEV1# % predicted Range 31–98 29–102 Mean±sd 64±21 65±25 Employment status In paid employment 11 (52) 4 (36) 7 (70) Student 4 (19) 2 (18) 2 (20) Retired due to age 1 (5) 1 (9) 0 Retired/left work due to PCD 4 (19) 3 (27) 1 (10) Carer for dependants 1 (5) 1 (9) 0 Other 0 0 0 Marital status Single 5 (24) 3 (27) 2 (20) Living with partner/spouse 16 (76) 8 (73) 8 (80) Separated from partner/spouse 0 0 Widowed 0 0 Fertility Conceived naturally 3 (27) 0 Conceived through IVF 1 (9) 2 (20) Infertility 3 (27) 2 (20) Not yet known 4 (36) 6 (60) Data are presented as n (%), unless otherwise stated. #: forced expiratory volume in 1 s (FEV1) is based on n=5 participants in the UK and n=8 in North America; data were unavailable for telephone interviews.

ved naturally 3 (27) 0 Conceived through IVF 1 (9) 2 (20) Infertility 3 (27) 2 (20) Not yet known 4 (36) 6 (60) Data are presented as n (%), unless otherwise stated. #: forced expiratory volume in 1 s (FEV1) is based on n=5 participants in the UK and n=8 in North America; data were unavailable for telephone interviews. TABLE 2 Participant quotes by topic

ved naturally 3 (27) 0 Conceived through IVF 1 (9) 2 (20) Infertility 3 (27) 2 (20) Not yet known 4 (36) 6 (60) Data are presented as n (%), unless otherwise stated. #: forced expiratory volume in 1 s (FEV1) is based on n=5 participants in the UK and n=8 in North America; data were unavailable for telephone interviews. TABLE 2 Participant quotes by topic Topic Quote Country of interviewee/sex/age band in years Impact of respiratory symptoms  “I had to tell the group not to worry because I start huffing and spluttering as I'm walking.” UK/female/36–50 “When I listen to myself breathe, I always wheeze.” USA/female/18–35 Impact of sinus symptoms “I'm always blowing my nose, doesn't matter what weather it is.” UK/female/36–50 “I always have to blow my nose before I eat if I wanna taste anything.” USA/female/36–50 Impact of ear symptoms/hearing loss “You have to ask people to repeat themselves so many times, they're just, like, ‘oh don't worry about it’.” UK/male/18–35 “I can't go white water rafting because I have tubes in my ears and my ears can't get wet.” USA/female/18–35 Impact of fertility issues “Finding out that I possibly can't have kids; that's when it started to panic me a little bit.” UK/male/18–35 “I'm still very uncertain if I ever wanna have children because I don't know how me having this illness will affect them.” USA/female/18–35 Impact of treatment burden “I don't really want to do it; it's kind of boring and it's not fun and I'd rather do something else. But obviously you have to do it.” UK/female/18–35 “I think it just requires more planning. I need to wake up earlier or start getting ready for bed earlier, I need to come home from work and do this; it's just more planning.” USA/female/18–35 Emotional functioning “I'm so frustrated with this illness, I just want it to go away, but, unfortunately, that's how I have to live.” UK/male/18–35 “…if you go to the doctor [and] you're feeling pretty good and you know your numbers are not good; that can be a big cause of anxiety.” USA/female/18–35 Social functioning “It has had such a huge impact on my life, and certainly I think it's contributed to the breakup of my first marriage.” UK/female/50–64 “…there have been times where I've had to cancel things because I've gotten sick. Getting sick can happen overnight; you're fine one day and the next day you feel awful.” USA/female/18–35

has had such a huge impact on my life, and certainly I think it's contributed to the breakup of my first marriage.” UK/female/50–64 “…there have been times where I've had to cancel things because I've gotten sick. Getting sick can happen overnight; you're fine one day and the next day you feel awful.” USA/female/18–35 Content analysis and item reduction Content analysis of the transcripts yielded the most important items for each of the 10 domains based on the frequency with which they were mentioned across adults. Saturation of content, across domains, was confirmed when no new themes emerged (figure 1). Our results indicated that this was achieved by the 5–7th interview, depending on the specific content area. We also harmonised the content across UK and North America to identify the most important topics. The items that were considered most important and relevant by the participants who performed the open-ended interviews were also scored as highly relevant by the 49 adult members of the PCD UK support group who completed the survey. Thus, content analysis of the interviews concurred with results of the survey (supplement table S). FIGURE 1 Saturation grids for UK and US participants: respiratory symptoms, sinus symptoms, and ear and hearing symptoms. Vertical lines indicate saturation was reached. All items above the horizontal lines were retained for the final questionnaire. Shaded cells refer to the first time the item was mentioned. #: participants did not indicate adverse effects of difficulty smelling; therefore it was not included in the final questionnaire.

Vertical lines indicate saturation was reached. All items above the horizontal lines were retained for the final questionnaire. Shaded cells refer to the first time the item was mentioned. #: participants did not indicate adverse effects of difficulty smelling; therefore it was not included in the final questionnaire. Cognitive testing Cognitive interviews were conducted with 15 adults (UK n=9, USA n=6). Review of these transcripts indicated that patients found the items clear, important and relevant and had no difficulty with the response options. Six items were added, based on patient input, after the cognitive testing phase (table 3). These topics included: making plans for the future (vacation, attending family events), treatment burden, intimacy, and pain associated with sinus disease. Thus, the final prototype instrument contained 48 items (QOL–PCD v1.2). Following this iterative process, the draft version of the QOL–PCD v.1.2 is ready to be tested in a psychometric validation study. TABLE 3 Summary of modifications to QOL–PCD after cognitive testing

intimacy, and pain associated with sinus disease. Thus, the final prototype instrument contained 48 items (QOL–PCD v1.2). Following this iterative process, the draft version of the QOL–PCD v.1.2 is ready to be tested in a psychometric validation study. TABLE 3 Summary of modifications to QOL–PCD after cognitive testing Modifications after cognitive testing Items added to scales Respiratory symptoms: Wheezing Chest tightness Sinus symptoms: Post-nasal drip Sinus pain Physical functioning: Carrying heavy things, such as books and shopping bags Health perceptions: I feel healthy Emotional functioning: Felt depressed Felt lonely Social functioning: Stay at home more often than would like Feel comfortable coughing in front of others Feel comfortable blowing nose in front of others Intimacy with a partner (kissing, hugging, sexual activity) Worried about being exposed to others who are sick Comfortable doing treatments (airway clearance, physiotherapy) in front of others Treatment burden Physiotherapy/airway clearance made you feel tired quickly Items deleted from scales Health perceptions: I feel in control of my PCD Emotional functioning: Felt angry Felt limited Felt self-conscious Social functioning Self-conscious coughing and blowing my nose in public Treatment burden: Treatments made you feel better Physiotherapy is hard work Wording modifications Emotional functioning: “Felt anxious” changed to “felt worried” “Felt frustrated” changed to “felt frustrated about doing your daily treatments”

ous Social functioning Self-conscious coughing and blowing my nose in public Treatment burden: Treatments made you feel better Physiotherapy is hard work Wording modifications Emotional functioning: “Felt anxious” changed to “felt worried” “Felt frustrated” changed to “felt frustrated about doing your daily treatments” Discussion This process, conducted in the UK and North America, yielded the first HRQoL instrument for adults with PCD, the QOL–PCD. It was developed following the guidelines published by the major regulatory bodies in Europe and the USA (i.e. EMEA and FDA) [13, 22, 23] and will be submitted to these agencies for consideration as an outcome measure for clinical trials. The most important principle governing its development was our reliance on patient input and their perspective at each phase. Thus, this tool systematically reflects how an adult with PCD “survives, feels, or functions” [13]. Given the rarity of this chronic disease, we developed the content cross-culturally in English-speaking countries (UK, USA and Canada) and found no discrepancies in content across countries. Additional input was obtained from the current literature and from medical experts across Europe and North America.

ctions” [13]. Given the rarity of this chronic disease, we developed the content cross-culturally in English-speaking countries (UK, USA and Canada) and found no discrepancies in content across countries. Additional input was obtained from the current literature and from medical experts across Europe and North America. Open-ended interviews highlighted the importance not only of patients' respiratory symptoms, but the effects of sinus disease and hearing problems on daily functioning. Although sinus disease is also problematic for patients with CF and non-CF bronchiectasis, patients with PCD emphasised the additional impact of their upper respiratory tract symptoms. This highlights that PCD has distinct features from other bronchiectatic diseases [26], and deserves individualised management. Thus, a number of items assessing rhino-sinus and ear symptoms appear on the final instrument, differentiating it from disease-specific HRQoL measures for adults with CF or non-CF bronchiectasis [12, 27].

hts that PCD has distinct features from other bronchiectatic diseases [26], and deserves individualised management. Thus, a number of items assessing rhino-sinus and ear symptoms appear on the final instrument, differentiating it from disease-specific HRQoL measures for adults with CF or non-CF bronchiectasis [12, 27]. The study population was not fully representative of the PCD population. In common with many previous studies, it was more difficult to recruit men than women with only three men (14%) participating in the interviews. Approximately 6% of patients with PCD have cardiovascular disease [5]. None of the 21 interviewees in this study had cardiovascular disease, but even if we had designed the study population to be representative of the PCD population we would have aimed to have only one patient. Moreover, we would not be able to recruit patients with the diverse spectrum of cardiac disease e.g. complex cyanotic heart disease versus simple cardiac anomaly. It is therefore a limitation of the questionnaire that it does not include items relevant to patients with cardiac disease.

e aimed to have only one patient. Moreover, we would not be able to recruit patients with the diverse spectrum of cardiac disease e.g. complex cyanotic heart disease versus simple cardiac anomaly. It is therefore a limitation of the questionnaire that it does not include items relevant to patients with cardiac disease. A reliable patient-reported outcome measure for PCD is particularly important, given that physiological measures such as forced expiratory volume in 1 s and LCI are not sensitive or predictive, and HRCT is not suitable for repeat testing due to radiation exposure. Importantly, the QOL–PCD provides a measure of the multidimensional effects of PCD on adults, from their own perspective including its impact on the upper and lower respiratory systems, treatment burden and social and emotional functioning. Reliability and validity studies are currently in process and will be reported in due course. In summary, the QOL–PCD was developed and has undergone cognitive testing in adults from several English-speaking countries. It has been translated into Dutch, German, and Danish, with plans to develop translations from major European countries and the Middle East. A multi-national, psychometric field-study is now planned to assess several forms of reliability and validity. Similar processes have been used to develop age-specific HRQoL measures for children and adolescents with PCD and parent caregivers. These instruments will also be translated into other languages and validated in future studies.

sychometric field-study is now planned to assess several forms of reliability and validity. Similar processes have been used to develop age-specific HRQoL measures for children and adolescents with PCD and parent caregivers. These instruments will also be translated into other languages and validated in future studies. Acknowledgements Research in Southampton is supported by NIHR Southampton Respiratory Biomedical Research Unit, NIHR Wellcome Trust Clinical Research Facility and AAIR Charity. Members of the PCD European Group provided expert opinion; JSL, MWL and LB are members of ERS Task Force for PCD Diagnostics (ERS TF-2014-04). JSL leads EU-funded COST Action BEAT-PCD (BM1407). Members of the PCD Support Group, UK (Chair Fiona Copeland) and PCD Foundation, North America (Director Michele Manion) contributed to all aspects of study conduct. This article has supplementary material available from erj.ersjournals.com Support statement: The research leading to these results has received funding from the European Union's Seventh Framework Programme under EC-GA No. 305404 BESTCILIA. In addition MRK and MWL receive grant support (U54HL096458) from the National Institutes of Health (NIH) through collaboration between the NIH Office of Rare Diseases Research (ORDR) at the National Center for Advancing Translational Science (NCATS), and the National Heart, Lung & Blood Institute (NHLBI).; AQ was supported from an Investigator-initiated grant, Gilead Sciences; the National PCD Service in Southampton UK is commissioned and funded by NHS England.

fice of Rare Diseases Research (ORDR) at the National Center for Advancing Translational Science (NCATS), and the National Heart, Lung & Blood Institute (NHLBI).; AQ was supported from an Investigator-initiated grant, Gilead Sciences; the National PCD Service in Southampton UK is commissioned and funded by NHS England. Conflict of interest: Disclosures can be found alongside the online version of this article at erj.ersjournals.com

Introduction Primary ciliary dyskinesia (PCD) is a rare heterogeneous disorder characterised by abnormal ciliary function and associated with abnormal ciliary ultrastructure in 70% of cases [1, 2]. Consequences of PCD include impaired mucociliary clearance of the airway causing upper and lower respiratory tract symptoms which usually present soon after birth. Neonatal manifestations range in severity from mild transient tachypnoea to significant respiratory failure requiring prolonged respiratory support [3–5]. Patients continue to have chronic, progressive symptoms of persistent wet cough and recurrent chest infections that almost invariably lead to bronchiectasis. Upper airway problems include rhinosinusitis and recurrent otitis media with hearing impairment [6]. Motile embryonic nodal cilia are important for left/right asymmetry; approximately half of PCD patients exhibit situs inversus and 6–12% heterotaxic syndromes which may be associated with complex congenital cardiac defects [7, 8]. Male Infertility is common since sperm flagella have a similar ultrastructure to cilia; the incidence of female infertility is less clear, but can be explained by immotile fallopian tube cilia [9].

bit situs inversus and 6–12% heterotaxic syndromes which may be associated with complex congenital cardiac defects [7, 8]. Male Infertility is common since sperm flagella have a similar ultrastructure to cilia; the incidence of female infertility is less clear, but can be explained by immotile fallopian tube cilia [9]. The prevalence, burden of disease and prognosis of PCD patients is difficult to determine due to limited representative international data. Reported prevalence varies from 1:2000 to 1:40 000, reflecting true variability as well as differences in access to diagnostic facilities [10–12]. A survey of 26 European countries found that PCD is underdiagnosed or diagnosed late particularly in countries with low healthcare expenditure [10]. In addition to expecting to improve respiratory prognosis [13–15], early diagnosis facilitates appropriate management; management is different to non-PCD-related serous otitis media, for example [16]. Diagnosis also allows genetic counselling for the family.

rticularly in countries with low healthcare expenditure [10]. In addition to expecting to improve respiratory prognosis [13–15], early diagnosis facilitates appropriate management; management is different to non-PCD-related serous otitis media, for example [16]. Diagnosis also allows genetic counselling for the family. There is currently no “gold standard” test to diagnose PCD [17]. European guidelines recommend that PCD should be confirmed in a specialist centre using appropriate diagnostic testing [18]. PCD diagnostic investigations are complex, requiring expensive infrastructure, and an experienced team of clinicians, scientists and microscopists [17, 19, 20]. Various models exist to deliver diagnostic services for this rare disease, generally with a network of satellite screening centres accessing a specialist centre [16, 20, 21]. The symptoms of PCD are nonspecific and secondary-care physicians need guidance of whom to refer for diagnostic testing [16]. To promote early diagnosis without overburdening specialist services, screening tools such as nasal nitric oxide (nNO) are used, which has been proved to be an efficient screening measures [21–23]. This, however, requires expensive equipment and trained technicians to obtain reliable measurements.

gnostic testing [16]. To promote early diagnosis without overburdening specialist services, screening tools such as nasal nitric oxide (nNO) are used, which has been proved to be an efficient screening measures [21–23]. This, however, requires expensive equipment and trained technicians to obtain reliable measurements. The aim of this study was to utilise easily available clinical information from a large prospective population to produce a scoring tool to predict whether symptomatic patients have PCD: PICADAR (PrImary CiliARy DyskinesiA Rule). We aimed to develop a tool that would be quick and easy to use by general respiratory and ear, nose and throat specialists. PICADAR's accuracy was externally validated in a second PCD diagnostic centre. Methods Ethics This research was approved by the National Research Ethics Service (NRES-06/Q1702/109). Study population Derivative group We analysed data from 641 consecutive patients with a definitive diagnostic outcome from the University Hospital Southampton (UHS) PCD diagnostic centre (2007–2013). A proforma was used to collect patient data, completed by a clinician through a clinical interview prior to diagnostic testing.

opulation Derivative group We analysed data from 641 consecutive patients with a definitive diagnostic outcome from the University Hospital Southampton (UHS) PCD diagnostic centre (2007–2013). A proforma was used to collect patient data, completed by a clinician through a clinical interview prior to diagnostic testing. External validation group We used data from a sample of 187 patients (93 PCD-positive and 94 PCD-negative) referred for testing to the Royal Brompton Hospital (RBH) to validate the score. An equal number of positive and negative referrals were randomly selected from the overall population of patients referred between 1983 and 2013. Using a similar protocol to UHS, a clinical history proforma was completed before diagnostic testing.

for testing to the Royal Brompton Hospital (RBH) to validate the score. An equal number of positive and negative referrals were randomly selected from the overall population of patients referred between 1983 and 2013. Using a similar protocol to UHS, a clinical history proforma was completed before diagnostic testing. Diagnostic testing The diagnostic criteria used in the UK (UHS and RBH) have previously been described in detail in Jackson et al. [24] and Lucas and Leigh [17]. In brief, a positive diagnosis is usually based on a typical clinical history with at least two abnormal diagnostic tests (“hallmark” transmission electron microscopy (TEM), “hallmark” ciliary beat pattern (CBP), nNO ≤30 nL·min−1). Occasionally patients with a strong history (e.g. sibling with PCD, “full” clinical phenotype (e.g. neonatal respiratory distress at term followed by daily wet cough, persistent rhinitis and glue ear)), are diagnosed based on either “hallmark” TEM or repeated high-speed video microscopy analysis (HSVMA) consistent with PCD. CBP was only considered positive if the pattern was typical of PCD rather than secondary ciliary dyskinesia either from two brushing biopsies or from one brushing biopsy with reanalysis following air–liquid interface culture.

r “hallmark” TEM or repeated high-speed video microscopy analysis (HSVMA) consistent with PCD. CBP was only considered positive if the pattern was typical of PCD rather than secondary ciliary dyskinesia either from two brushing biopsies or from one brushing biopsy with reanalysis following air–liquid interface culture. Clinical data Data was collected on sex, date of birth, age at assessment and ethnicity. Neonatal data collected included admittance to a special care babies unit, neonatal respiratory support, neonatal rhinitis or chest symptoms. Data on the presence of situs abnormalities, congenital cardiac defect, chronic (>3 months) cough, rhinitis, sinusitis, ear problems, history of pneumonia and bronchiectasis was collated. Family history of PCD, bronchiectasis, hearing problems, asthma and consanguinity were included. Data on clinical history was coded as yes=0, no=1 or missing=99. For the adult population, subfertility was recorded if the patient had difficultly conceiving but had children, used in vitro fertilisation or if they stated they were never able to conceive. Model development Potential predictors were restricted to information readily available in a nonspecialist setting. From the derivation group, 27 potential variables were identified. Two-tailed parametric (t-test) or nonparametric (Mann–Whitney) tests, Chi-squared test or Fisher's exact test (as appropriate) were used to compare the characteristics of positive and negative referrals.

ion readily available in a nonspecialist setting. From the derivation group, 27 potential variables were identified. Two-tailed parametric (t-test) or nonparametric (Mann–Whitney) tests, Chi-squared test or Fisher's exact test (as appropriate) were used to compare the characteristics of positive and negative referrals. Logistic regression analysis was used to develop a simplified practical prediction tool. First, potential predictors were entered into the model individually using forward step-wise methods. This allowed us to identify and select the significant predictors for PCD and assess their influence on a positive PCD diagnosis. Sensitivity, specificity and overall accuracy (the weighted average of the models sensitivity and specific) from selected significant predictors were interpreted [25].

ard step-wise methods. This allowed us to identify and select the significant predictors for PCD and assess their influence on a positive PCD diagnosis. Sensitivity, specificity and overall accuracy (the weighted average of the models sensitivity and specific) from selected significant predictors were interpreted [25]. Model performance The model's ability to discriminate between those with and without PCD was assessed by plotting the receiver operating characteristic (ROC) curve and calculating the area under the ROC curve (AUC). Discrimination was considered moderate if AUC 0.6–0.8 and good if AUC >0.8 [26]. The Hosmer–Lemeshow goodness-of-fit-test was used to assess the calibration of the model, i.e. how well the predicted probabilities agreed with the prevalence of the outcome in patient subgroups. A Hosmer–Lemeshow goodness-of-fit-test [27] result of <0.05 indicates that the predicted probabilities and the actual outcome agree poorly [28]. Subjects with missing data were excluded on a case-wise basis; however, to confirm the model's accuracy, multiple imputation was used to check for any biases that can occur in complete case analysis along with a substantial loss of power and precision [29, 30].

ed probabilities and the actual outcome agree poorly [28]. Subjects with missing data were excluded on a case-wise basis; however, to confirm the model's accuracy, multiple imputation was used to check for any biases that can occur in complete case analysis along with a substantial loss of power and precision [29, 30]. Clinical prediction tool The best model from the logistic regression allowed for the calculation of a diagnostic predictive tool (PICADAR) to estimate the probability of a positive PCD diagnosis based on total score. The score for each predictor corresponds to their regression coefficient rounded to the nearest integer. A ROC curve was plotted to assess the predictive performance of PICADAR for comparison with the original model. Each score has a corresponding accuracy (i.e. sensitivity and specificity) of predicting a positive or negative diagnosis. Validation in external population The discriminative ability of the scores in the validation population was assessed using ROC curve analysis. All analyses were performed by using SPSS Statistics for Windows version 21.0 (IBM, Armonk, NY, USA). Results Study population Of 641 consecutive participants in the derivation group, 75 (12%) were diagnosed with PCD and 566 (88%) had a negative diagnosis. Median (range) age at assessment was 9 (0–79) years and 44% of patients were male.

Validation in external population The discriminative ability of the scores in the validation population was assessed using ROC curve analysis. All analyses were performed by using SPSS Statistics for Windows version 21.0 (IBM, Armonk, NY, USA). Results Study population Of 641 consecutive participants in the derivation group, 75 (12%) were diagnosed with PCD and 566 (88%) had a negative diagnosis. Median (range) age at assessment was 9 (0–79) years and 44% of patients were male. The validation group was selected to include similar numbers of positive and negative diagnoses. The participants were younger than the derivation group; they were also more likely to be non-white and from a consanguineous background, reflecting the different populations served by UHS and RBH (table 1). TABLE 1 Demographical characteristics of the two study populations: the derivation group and the validation group#

e participants were younger than the derivation group; they were also more likely to be non-white and from a consanguineous background, reflecting the different populations served by UHS and RBH (table 1). TABLE 1 Demographical characteristics of the two study populations: the derivation group and the validation group# Derivation group Validation group p-value Total PCD-positive PCD-negative Total PCD-positive PCD-negative Subjects 641 75 566 157 80 77 Age at assessment years 9 (0–79) 6 (0–67) 9 (0–79) 3 (0–18) 6 (0–17) 2 (0–12) <0.001 Male 283 (44) 34 (45) 249 (44) 78 (50) 38 (48) 40 (52) 0.211 Gestational age months 39±2.6 39.6±1.6 38.9±2.7 39±2.3 39±2.0 39±2.5 0.332 Pre-term 87 (14) 7 (9) 80 (14) 17 (11) 7 (9) 10 (13) 0.357 Siblings with PCD 27 (4) 18 (24) 9 (2) 27 (17) 24 (30) 3 (4) <0.001 Other family with PCD 10 (2) 4 (5) 6 (1) 8 (5) 7 (9) 1 (1) 0.007 Consanguinity 20 (3) 12 (16) 8 (1) 38 (24) 35 (44) 3 (4) <0.001 Ethnicity White 482 (75) 57 (76) 425 (75) 90 (57) 31 (39) 59 (77) <0.001 Other 55 (9) 17 (23) 38 (7) 57 (36) 39 (49) 18 (23) <0.001 Not stated 104 (16) 1 (1) 103 (18) 10 (7) 10 (12) 0 (0) 0.002 Data are presented as n, median (range), mean±sd or n (%), unless otherwise stated. PCD: primary ciliary dyskinesia. #: missing values not presented.

) 57 (76) 425 (75) 90 (57) 31 (39) 59 (77) <0.001 Other 55 (9) 17 (23) 38 (7) 57 (36) 39 (49) 18 (23) <0.001 Not stated 104 (16) 1 (1) 103 (18) 10 (7) 10 (12) 0 (0) 0.002 Data are presented as n, median (range), mean±sd or n (%), unless otherwise stated. PCD: primary ciliary dyskinesia. #: missing values not presented. Clinical and family characteristics Both PCD-positive and PCD-negative groups had a high prevalence of a persistent daily wet cough throughout life (PCD-positive 93.3%, PCD-negative 85.1%, p=0.069). PCD-positive patients were more likely to report neonatal problems requiring admittance to a neonatal unit (61.3%, 13.6%, p<0.001), neonatal rhinitis (26.6%, 6.5%, p<0.001) and neonatal chest symptoms (e.g. wet cough, tachypnoea, oxygen requirement) (74.6%, 17.1%, p<0.001). Symptoms were higher among the PCD-positive group for persistent perennial rhinitis (81.3%, 57.4%, p<0.001), serous otitis media (glue ear) (57.3%, 19.2%, p<0.001) and hearing loss (49.3%, 15.9%, p<0.001). Situs abnormalities (44.0%, 3.9%, p<0.001) and congenital cardiac defects (8.0%, 1.7%, p=0.001) were also more common in the PCD-positive group. Fertility data was available for 152 referrals, with a significantly higher percentage reporting subfertility in the PCD-positive group (90%, 18.4%, p<0.001). Family history of PCD in siblings (24%, 1.6%, p<0.001) or in other family members (5.3%, 1.1%, p=0.012) was significantly higher among the PCD-positive group. Consanguinity (16.0%, 1.4%, p<0.001) was more common in PCD-positive group. Clinical characteristics of the derivation group are summarised in table 2 and of the validation group in online supplementary table E1. TABLE 2 Clinical symptom characteristics of the derivation group

antly higher among the PCD-positive group. Consanguinity (16.0%, 1.4%, p<0.001) was more common in PCD-positive group. Clinical characteristics of the derivation group are summarised in table 2 and of the validation group in online supplementary table E1. TABLE 2 Clinical symptom characteristics of the derivation group Total PCD-positive PCD-negative Odds ratio (95% CI) p-value Subjects 641 75 566 Neonatal symptoms Neonatal respiratory support 72 (11.2) 31 (41.3) 41 (7.2) 9.77 (5.53–17.26) <0.001 Neonatal chest symptoms 153 (23.0) 56 (74.6) 97 (17.1) 13.56 (7.60–24.11) <0.001 Neonatal rhinitis 57 (8.9) 20 (26.6) 37 (6.5) 5.53 (2.99–10.23) <0.001 Respiratory symptoms Persistent daily wet cough 552 (86.1) 70 (93.3) 482 (85.1) 2.38 (0.93–6.07) 0.069 Recurrent wheeze 254 (39.6) 36 (48.0) 218 (38.5) 1.39 (0.86–2.26) 0.176 Previous pneumonia 227 (35.4) 31 (41.3) 196 (34.6) 1.14 (0.69–1.88) 0.585 Bronchiectasis 202 (31.5) 22 (29.3) 180 (31.8) 0.94 (0.54–1.61) 0.83 Upper airway and ear symptoms Perennial persistent rhinitis 386 (60.2) 61 (81.3) 325 (57.4) 3.20 (1.75–5.86) <0.001 Chronic sinusitis 159 (24.8) 21 (28.0) 138 (24.3) 1.19 (0.69–2.05) 0.52 Hearing loss 127 (19.8) 37 (49.3) 90 (15.9) 5.90 (3.52–9.98) <0.001 Chronic acute otitis media 165 (25.7) 25 (33.3) 140 (24.7) 1.41 (0.85–2.32) 0.117 Serous otitis media 152 (23.7) 43 (57.3) 109 (19.2) 3.24 (2.11–4.96) <0.001 Chronic ear perforation 59 (9.2) 9 (12.0) 50 (8.8) 5.9 (3.52–9.98) 0.398 Ear surgery 105 (16.3) 24 (32.0) 81 (14.3) 2.81 (1.64–2.83) <0.001 Other clinical characteristics Neonatal unit 123 (19.2) 46 (61.3) 77 (13.6) 11.13 (6.46– 19.17) <0.001 Situs abnormality 55 (8.5) 33 (44.0) 22 (3.9) 17.19 (9.18–32.19) <0.001 Congenital cardiac defect 16 (2.5) 6 (8.0) 10 (1.7) 4.75 (1.81–12.48) 0.001 Hydrocephalus 7 (1.1) 1 (1.3) 6 (1.0) 1.14 (0.13–9.61) 0.903 Developmental delay# 43 (6.7) 8 (10.6) 35 (6.2) 1.68 (0.75–3.82) 0.197 Family history of disease PCD in siblings 27 (4.2) 18 (24.0) 9 (1.6) 19.23 (8.24–44.87) <0.001 PCD in extended family 10 (1.5) 4 (5.3) 6 (1.1) 5.22 (1.44–18.98) 0.012 Asthma 206 (32.1) 12 (16.0) 194 (34.2) 0.35 (0.18–0.67) 0.002 Bronchiectasis 28 (4.3) 3 (4.0) 25 (4.4) 0.99 (0.29–3.40) 0.998 Otitis media 65 (10.1) 5 (6.6) 60 (10.6) 0.69 (0.26–1.80) 0.456 Subfertility¶ 36 (23.6) 10 (90) 26 (18.4) 44.23 (5.42–360.91) <0.001 Data are presented as n or n (%), unless otherwise stated. PCD: primary ciliary dyskinesia.

4 (34.2) 0.35 (0.18–0.67) 0.002 Bronchiectasis 28 (4.3) 3 (4.0) 25 (4.4) 0.99 (0.29–3.40) 0.998 Otitis media 65 (10.1) 5 (6.6) 60 (10.6) 0.69 (0.26–1.80) 0.456 Subfertility¶ 36 (23.6) 10 (90) 26 (18.4) 44.23 (5.42–360.91) <0.001 Data are presented as n or n (%), unless otherwise stated. PCD: primary ciliary dyskinesia. #: developmental delay includes those who present with gross motor delay, social delay or language delay; ¶: subfertility is based on a total of 152 referrals (positive referrals n=11, negative referrals n=141). Development of PICADAR score Of the 27 binary variables considered for selection, the best logistic regression model included seven significant predictors. In order of importance (based on their corresponding odds ratio) these predictors were situs inversus, birth at full term, neonatal chest symptoms, admission to a neonatal unit, congenital cardiac defect, rhinitis, and ear and hearing symptoms (table 3). Similar results were found when multiple imputation was applied (online supplementary table E2). The overall accuracy of this model was 90%, and the sensitivity and specificity were 71% and 94%, respectively. The discriminant ability (AUC) of this model was 0.92 (figure 1). The Hosmer–Lemeshow test showed good agreement between the predicted probabilities and the actual outcome (p=0.64). FIGURE 1 PICADAR: receiver operating characteristic (ROC) curves for the best prediction model (area under the ROC curve (AUC) 0.92, 95% CI 0.87–0.95) and the predication tool (AUC 0.91, 95% CI 0.87–0.95) in the derivation group.

showed good agreement between the predicted probabilities and the actual outcome (p=0.64). FIGURE 1 PICADAR: receiver operating characteristic (ROC) curves for the best prediction model (area under the ROC curve (AUC) 0.92, 95% CI 0.87–0.95) and the predication tool (AUC 0.91, 95% CI 0.87–0.95) in the derivation group. TABLE 3 Factors for the prediction of primary ciliary dyskinesia selected by step-wise logistic regression Regression coefficient Odds ratio (95% CI) p-value Simplified regression coefficient tool# Situs inversus 3.54 34.48 (11.6–101.8) <0.001 4 Gestational age (full term) 2.20 9.06 (2.9–27.4) <0.001 2 Neonatal chest symptoms 1.91 6.79 (2.7–16.7) <0.001 2 Neonatal unit 1.90 6.70 (2.7–16.3) <0.001 2 Congenital cardiac defect 1.57 4.83 (1.1–22.2) 0.043 2 Rhinitis 1.22 3.40 (1.2–8.9) 0.013 1 Ear and hearing symptoms 0.95 2.59 (1.2–5.8) 0.021 1 #: regression coefficients of the main model are rounded to the nearest integer.

natal chest symptoms 1.91 6.79 (2.7–16.7) <0.001 2 Neonatal unit 1.90 6.70 (2.7–16.3) <0.001 2 Congenital cardiac defect 1.57 4.83 (1.1–22.2) 0.043 2 Rhinitis 1.22 3.40 (1.2–8.9) 0.013 1 Ear and hearing symptoms 0.95 2.59 (1.2–5.8) 0.021 1 #: regression coefficients of the main model are rounded to the nearest integer. PICADAR (figure 2) was designed as an easily scored predictive tool based on the seven-variable-predictor model. The presence of each clinical factor contributed to the total score following adjustment of its regression coefficient values to an integer between 1 and 4. This adjustment had little effect on discriminative ability (model AUC 0.92, PICADAR AUC 0.91) (figure 1). FIGURE 2 PICADAR is a predictive score with seven simple questions to predict the likelihood of having primary ciliary dyskinesia (PCD). It can be used in any patients with chronic respiratory symptoms starting in early childhood. The total score is calculated and the individual probability of having PCD diagnosis can be estimated from the probability curve shown in figure 3. FIGURE 3 PICADAR: probability curve. Once the total PICADAR score is calculated from figure 2, the individual probability of having a primary ciliary dyskinesia diagnosis is estimated from the probability curve.

PICADAR (figure 2) was designed as an easily scored predictive tool based on the seven-variable-predictor model. The presence of each clinical factor contributed to the total score following adjustment of its regression coefficient values to an integer between 1 and 4. This adjustment had little effect on discriminative ability (model AUC 0.92, PICADAR AUC 0.91) (figure 1). FIGURE 2 PICADAR is a predictive score with seven simple questions to predict the likelihood of having primary ciliary dyskinesia (PCD). It can be used in any patients with chronic respiratory symptoms starting in early childhood. The total score is calculated and the individual probability of having PCD diagnosis can be estimated from the probability curve shown in figure 3. FIGURE 3 PICADAR: probability curve. Once the total PICADAR score is calculated from figure 2, the individual probability of having a primary ciliary dyskinesia diagnosis is estimated from the probability curve. The likelihood of a patient having PCD can be estimated by comparing their score to the probability curve (figure 3) or cut-offs could be used. The highest combined sensitivity and specificity (0.90 and 0.75, respectively) was at the cut-off value of 5 points (table 4). The maximum PICADAR score was 14. This corresponded to a 99.80% probability of having PCD, a score ≥10 had a probability of 92.6% and a score ≥5 had a probability of 11.10% (figure 3 and online supplementary table E3). In the UHS derivation group, of the PCD-positive, 6.0% had scores ≤5, 58.0% had scores 6–9 and 36.0% had scores ≥10. In the PCD-negative group, 79.4% had scores ≤5, 20.2% had scores 6–9 and only 0.4% had scores ≥10 (table 5). TABLE 4 Performance measures including sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PICADAR prediction tool for different cut-off values calculated from the derivation group and the validation group

≤5, 20.2% had scores 6–9 and only 0.4% had scores ≥10 (table 5). TABLE 4 Performance measures including sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PICADAR prediction tool for different cut-off values calculated from the derivation group and the validation group Cut-off score Derivation group Validation group Sensitivity Specificity PPV NPV Sensitivity Specificity PPV NPV 0 >0.99 <0.01 0.12 >0.99 <0.01 0.51 0.00 1 >0.99 0.01 0.12 1.0 2 >0.99 0.04 0.12 1.0 >0.99 0.01 0.51 1.00 3 0.97 0.20 0.14 0.98 0.99 0.35 0.61 0.97 4 0.97 0.48 0.20 0.99 0.93 0.63 0.72 0.90 5 0.90 0.75 0.32 0.98 0.86 0.73 0.77 0.83 6 0.89 0.83 0.41 0.98 0.81 0.76 0.78 0.79 7 0.76 0.94 0.63 0.97 0.73 0.89 0.79 0.79 8 0.63 0.96 0.68 0.95 0.53 0.90 0.85 0.65 9 0.34 0.99 0.82 0.92 0.35 0.96 0.90 0.59 10 0.31 >0.99 0.80 0.92 0.29 0.96 0.88 0.57 11 0.19 >0.99 0.72 0.90 0.25 0.99 0.96 0.56 12 0.13 >0.99 1.0 0.90 0.13 0.99 0.93 0.52 13 0.01 >0.99 1.0 0.88 0.05 >0.99 1.00 0.50 14 <0.01 >0.99 0.88 0.01 >0.99 1.00 0.49 TABLE 5 The distribution of scores (≤5, 6–9 and ≥10) in primary ciliary dyskinesia (PCD) positive and PCD-negative participants using PICADAR in the derivation group (n=288) and in the validation group (n=157) (only children <18 years included) Derivation group Validation group PCD-positive PCD-negative PCD-positive PCD-negative Subjects 50 238 79 78 ≤5 3 (6.0) 189 (79.4) 15 (18.7) 59 (75.6) 6–9 29 (58.0) 48 (20.2) 42 (53.3) 16 (20.5) ≥10 18 (36.0) 1 (0.4) 22 (28.0) 3 (3.8) Data are presented as n or n (%).

TABLE 5 The distribution of scores (≤5, 6–9 and ≥10) in primary ciliary dyskinesia (PCD) positive and PCD-negative participants using PICADAR in the derivation group (n=288) and in the validation group (n=157) (only children <18 years included) Derivation group Validation group PCD-positive PCD-negative PCD-positive PCD-negative Subjects 50 238 79 78 ≤5 3 (6.0) 189 (79.4) 15 (18.7) 59 (75.6) 6–9 29 (58.0) 48 (20.2) 42 (53.3) 16 (20.5) ≥10 18 (36.0) 1 (0.4) 22 (28.0) 3 (3.8) Data are presented as n or n (%). Validation of the PICADAR score Validation of PICADAR used data from an independent set of 93 randomly selected PCD-positive patients and 94 PCD-negative patients from RBH. Data for all PICADAR predictors were available in 157 (84%) out of 187 of the validation group, all of whom were <18 years of age. The remaining 30 were excluded due to missing symptom data. Positive cases accounted for 79 (50%) of the population, 79 (50%) were male; mean age at assessment was 4 years. Scores in the validation group ranged from 0 to 14 (mean±sd 5.9±3.3). The mean±sd PICADAR score was higher in the PCD-positive group (PCD-positive 7.9±2.8, PCD-negative 3.8±2.3; p<0.01) and distribution of the scores differed between groups (online supplementary figure E1). ROC curve analysis confirmed the performance of the score with AUC 0.87 (95% CI 0.81–0.94) (figure 4). In the validation group, 18.7% of the PCD-positive group had a score ≤5, 53.3% had a score 6–9 and 29.1% had a score ≥10. For the PCD-negative group, 75.6% has a score ≤5, 20.5% had a score of 6–9 and 3.8% had a score ≥10 (table 5). FIGURE 4 Receiver operating characteristic (ROC) curve for PICADAR (area under the ROC curve 0.87, 95% CI 0.81–0.94) in the validation group.

up had a score ≤5, 53.3% had a score 6–9 and 29.1% had a score ≥10. For the PCD-negative group, 75.6% has a score ≤5, 20.5% had a score of 6–9 and 3.8% had a score ≥10 (table 5). FIGURE 4 Receiver operating characteristic (ROC) curve for PICADAR (area under the ROC curve 0.87, 95% CI 0.81–0.94) in the validation group. A second predictive tool, PICADAR+S, which includes the variable “siblings with PCD” was developed and validated for patients with one of more siblings. When validated, this tool was also shown to discriminate between positive and negative referrals (AUC 0.94, 95% CI 0.90–0.97) (see online supplementary material for full description).

ive tool, PICADAR+S, which includes the variable “siblings with PCD” was developed and validated for patients with one of more siblings. When validated, this tool was also shown to discriminate between positive and negative referrals (AUC 0.94, 95% CI 0.90–0.97) (see online supplementary material for full description). Discussion Statement of principal findings We have developed an easy-to-use predictive score for determining the likelihood of an individual having a diagnosis of PCD. The score accurately predicts a positive or negative test result in patients with daily lower respiratory tract symptoms throughout life. PICADAR was developed and validated in patients referred to specialist diagnostic centres; at this stage we would not aim for it to be used in primary care, but anticipate that it could be used by respiratory centres to guide referral to specialist PCD centres. PICADAR should raise awareness of symptoms associated with PCD, and stimulate discussion and research to further refine the tool. PCD centres could use PICADAR to identify patients who should be investigated further following inconclusive or equivocal PCD tests. In resource limited countries with no diagnostic facilities the tool could be used to attach a PCD-likelihood to the patients; this is important for international research registries and metacohorts. There is no gold standard test for PCD, and testing is restricted to centres with the infrastructure and expertise to analyse and interpret HSVMA or TEM images and genotype data [17, 20, 31].

Discussion Statement of principal findings We have developed an easy-to-use predictive score for determining the likelihood of an individual having a diagnosis of PCD. The score accurately predicts a positive or negative test result in patients with daily lower respiratory tract symptoms throughout life. PICADAR was developed and validated in patients referred to specialist diagnostic centres; at this stage we would not aim for it to be used in primary care, but anticipate that it could be used by respiratory centres to guide referral to specialist PCD centres. PICADAR should raise awareness of symptoms associated with PCD, and stimulate discussion and research to further refine the tool. PCD centres could use PICADAR to identify patients who should be investigated further following inconclusive or equivocal PCD tests. In resource limited countries with no diagnostic facilities the tool could be used to attach a PCD-likelihood to the patients; this is important for international research registries and metacohorts. There is no gold standard test for PCD, and testing is restricted to centres with the infrastructure and expertise to analyse and interpret HSVMA or TEM images and genotype data [17, 20, 31]. Measurement of nNO provides a good screening tool [21, 23] to differentiate PCD-positive and non-PCD in patients with symptoms. In the study population which contributed to the development of PICADAR, we previously reported in Jackson et al. [24] that a cut-off of 30 nL·min−1 was both sensitive (0.91, 95% CI 0.76–0.98) and specific (0.96, 95% CI 0.93–0.98); of 301 consecutive referrals for diagnostic testing including nNO, 31 (91%) out of 34 of PCD-positive patients had low nNO (true positive), 10 (3%) out of 267 of PCD-negative patients had low nNO (false positive) and 3/34 (9%) of PCD-positives had nNO >30 nL·min−1 (false negative). However, the equipment for measuring nNO is not widely available outside specialist centres and needs trained technicians to obtain reliable readings. We designed PICADAR for use outside specialist diagnostic centres. Our data suggests similar accuracy in comparison with nNO, e.g. a cut-off score of ≥5 using PICADAR had 90% sensitivity and 75% specificity to differentiate PCD-positive and PCD-negative patients in the derivation group, and had 86% sensitivity and 73% specificity in the validation group. If patients with a score ≤4 had not proceeded to further testing, 167 (70.2%) and 57 (73.1%) negative patients would have avoided formal testing at the diagnostic centres; however, two (4.0%) positive patients in the derivation group and 11 (13.9%) in the validation group would have been missed (table 5). The tool therefore should not be used in isolation when deciding who to refer. We believe that promotion of the tool is likely to raise awareness of the most common symptoms associated with PCD. Although PICADAR will “miss” some patients when used in isolation, since diagnosis is currently commonly missed due to lack of physician awareness, widespread use of PICADAR would inevitably increase the number of actual diagnoses.

f the tool is likely to raise awareness of the most common symptoms associated with PCD. Although PICADAR will “miss” some patients when used in isolation, since diagnosis is currently commonly missed due to lack of physician awareness, widespread use of PICADAR would inevitably increase the number of actual diagnoses. Strengths and limitations of the study PICADAR comprises seven predictive variables including full-term gestational age, admittance to a neonatal unit, neonatal chest symptoms, persistent perennial rhinitis, chronic ear and hearing symptoms, situs abnormalities, and presence of a cardiac defect; such items are easily ascertained and quick to compute in any clinical setting. We did not specify cardiac defects associated with laterality defects within the score because we want PICADAR to be used by nonspecialists. PICADAR was derived in a specialist PCD centre (UHS) and validated externally in another centre (RBH). Although these two diagnostic centres are both situated in Southern England, they have different demographic populations in terms of ethnicity, consanguinity and age at assessment. Good discriminant ability was maintained when used in the validation group with AUC 0.87. The process of developing a clinical prediction rule includes four stages before ever being implemented in routine practice (derivation, internal validation, external validation and impact analysis). If an external validation is done directly in another setting, internal validation is not necessary. Therefore, as of now, we have completed all three of the requested stages of the rule development. Once these results are published, further validations can be done in other countries and settings, by other investigators, so that they can provide additional evidence for its validity for further implementation in practice [32].

e, as of now, we have completed all three of the requested stages of the rule development. Once these results are published, further validations can be done in other countries and settings, by other investigators, so that they can provide additional evidence for its validity for further implementation in practice [32]. PICADAR was developed in a large clinically relevant population. Consecutive patients with a diagnostic outcome were included. Patients had been referred based on symptoms and/or family history. Diagnosis was based on a combination of tests including nNO measurements, HSVMA to assess ciliary function and TEM to assess ciliary ultrastructure [16, 18]. A detailed clinical proforma was completed by health professionals before diagnostic testing was started, thus reducing bias. Using PICADAR, patients with a score ≥10 had a >90% probability of testing positive for PCD. Those with a score ≥5 had a >11% chance of being diagnosed PCD-positive (figure 3 and online supplementary table E3). We believe that this guidance will support appropriate referrals of patients for specialist testing, particularly where patients are geographically remote from a diagnostic centre. Clinicians using the score need to be aware that individuals with low risk scores might still have PCD (table 5). It should be noted that persistent wet cough is not included in the score because virtually all positive and negative referrals had chronic cough; therefore, the score is for use in patients with chronic cough as a precondition.

Using PICADAR, patients with a score ≥10 had a >90% probability of testing positive for PCD. Those with a score ≥5 had a >11% chance of being diagnosed PCD-positive (figure 3 and online supplementary table E3). We believe that this guidance will support appropriate referrals of patients for specialist testing, particularly where patients are geographically remote from a diagnostic centre. Clinicians using the score need to be aware that individuals with low risk scores might still have PCD (table 5). It should be noted that persistent wet cough is not included in the score because virtually all positive and negative referrals had chronic cough; therefore, the score is for use in patients with chronic cough as a precondition. A potential limitation is that a significant amount of data was missing for some variables. For example, a large proportion of the adult population did not know their gestational age and none of the children would yet know their fertility status. Complete case analysis was used to deal with missing data; however, this can lead to bias. To overcome this obstacle, multiple imputation was used to replace missing values within the model's significant variables [29, 30]. The pooled result obtained from five imputed datasets showed the best model is accurate (online supplementary table E2). Importantly, it must also be emphasised that PICADAR was developed using a population already referred for diagnostic testing. It was developed with the aim of identifying appropriate patients for referral from secondary care and will now require validation in this setting. Finally, patients with equivocal results were excluded from the derivation and validation populations. This may artificially improve the tool's performance; however, good discrimination was found when the tool was validated in a second diagnostic centre.

m secondary care and will now require validation in this setting. Finally, patients with equivocal results were excluded from the derivation and validation populations. This may artificially improve the tool's performance; however, good discrimination was found when the tool was validated in a second diagnostic centre. Future research The scores were derived using combined data from adults and children, but we expect that separate scores for adults and children might further improve accuracy. We therefore propose further research in large cohorts of children and adults to derive separate scoring systems. PICADAR includes a number of predictors based in early life, including gestational age and neonatal chest symptoms, that may be difficult to recall in adulthood. Similarly, subfertility was more common in the PCD-positive group and is likely to be a strong predictor for adult diagnoses. While the derivation group consisted of a wide range of age groups, the majority of referrals in the external validation group were children. Furthermore, the scores were developed and validated in UK specialist diagnostic centres, but referrals originate from nonspecialist services, and therefore future validation will be needed in referral centres and in centres outside of the UK.

s, the majority of referrals in the external validation group were children. Furthermore, the scores were developed and validated in UK specialist diagnostic centres, but referrals originate from nonspecialist services, and therefore future validation will be needed in referral centres and in centres outside of the UK. Conclusions PICADAR provides the first validated tool to aid appropriate referral of patients for diagnostic testing. It was designed to be easily applied in a nonspecialist setting to determine which patients with chronic chest symptoms require PCD diagnostic testing. PICADAR is a simple cost-effective score suitable for use in all clinical settings. The tools are now available for validation in a variety of clinical settings. Acknowledgements Gary Connett, Julian Legg (Dept of Paediatrics, Southampton University Hospitals National Health Service Trust, Southampton, UK) and Karen Lock (Primary Ciliary Dyskinesia Centre, UHS, Southampton, UK) undertook clinical assessments. Claire Jackson, Patricia Goggin, Elizabeth Adam and Janice Cole (Primary Ciliary Dyskinesia Centre, UHS, Southampton, UK) undertook diagnostic analyses. Michael Leshen and Alyson Roberts (Primary Ciliary Dyskinesia Centre, Dept of Paediatrics, RBH, London, UK) helped access clinical data. This article has supplementary material available from erj.ersjournals.com

Acknowledgements Gary Connett, Julian Legg (Dept of Paediatrics, Southampton University Hospitals National Health Service Trust, Southampton, UK) and Karen Lock (Primary Ciliary Dyskinesia Centre, UHS, Southampton, UK) undertook clinical assessments. Claire Jackson, Patricia Goggin, Elizabeth Adam and Janice Cole (Primary Ciliary Dyskinesia Centre, UHS, Southampton, UK) undertook diagnostic analyses. Michael Leshen and Alyson Roberts (Primary Ciliary Dyskinesia Centre, Dept of Paediatrics, RBH, London, UK) helped access clinical data. This article has supplementary material available from erj.ersjournals.com Support statement: The National PCD Diagnostic Services at University Hospital Southampton (UHS) and Royal Brompton Hospital London (RBH) are commissioned and funded by NHS England. PCD research at UHS, Bern and RBH receives research funding from the European Union's Seventh Framework Programme under EC-GA no. 305404 BESTCILIA: Better Experimental Screening and Treatment for Primary Ciliary Dyskinesia. The researchers are supported by the network of COST Action BEAT-PCD: Better Evidence to Advance Therapeutic options for PCD (BM 1407). J.S. Lucas, C. Hogg, M. Goutaki, C. Kuehni and L. Behan are members of the European Respiratory Society PCD Taskforce for PCD Diagnostics (ERS TF-2014-04). PCD research in Southampton is supported by NIHR Southampton Respiratory Biomedical Research Unit and NIHR Wellcome Trust Clinical Research Facility. Conflict of interest: None declared.

Introduction Recent advances in the diagnosis of patients with primary ciliary dyskinesia (PCD) have included networks of specialists developing protocol-driven testing [1–4], international consensus guidelines [5] and rapid expansion of known PCD-related genes [3]. There is no “gold-standard” test for PCD, hence European consensus guidelines (2009) [5] recommend a combination of tests including nasal nitric oxide (nNO) screening [4, 6], high-speed video microscopy analysis (HSVMA) of ciliary beat frequency (CBF) and pattern (CBP) [7–10] and transmission electron microscopy (TEM) analysis of ciliary ultrastructure [11, 12]. Reanalysis following submerged [13] or air–liquid interface (ALI) [14] culture may be useful to exclude secondary ciliary dyskinesia or confirm PCD when analysis of the primary sample is abnormal, and may provide additional cilia if the primary sample is inadequate. The 2009 guidelines [5] also suggest potential adjuncts to diagnosis including immunofluorescence labelling of cilia proteins [15], pulmonary radioaerosol mucociliary clearance [16, 17] and genotyping. Since 2009 there have been rapid advances in the discovery of genes responsible for PCD [1, 3], thus allowing genetic testing to take a prominent position in some countries, but it is currently not funded in the English public healthcare system. The English PCD service [1, 18] diagnoses PCD using nNO, HSVMA and TEM, with reanalysis following ALI culture for inconclusive and positive samples.

sible for PCD [1, 3], thus allowing genetic testing to take a prominent position in some countries, but it is currently not funded in the English public healthcare system. The English PCD service [1, 18] diagnoses PCD using nNO, HSVMA and TEM, with reanalysis following ALI culture for inconclusive and positive samples. Several articles have reported the accuracy of individual tests for the diagnosis of PCD, but none have considered all available diagnostic data. Most reports have failed to include the significant numbers of “inconclusive” results [19]. The aim of this study was to determine the accuracy of PCD diagnostic tests (nNO, HSVMA and TEM) when used singularly or in combination, based on a large prospective study of consecutive patients referred for diagnostic testing. Methods Local and national research and development and ethical approvals were obtained (Southampton and South West Hampshire research ethics 07/Q1702/109). Participants 868 consecutive subjects were referred to the national PCD centre at University Hospital Southampton (UHS) for diagnostic testing between 2007 and 2013; 654 had adequate data and samples for inclusion. The population served by the centre is predominantly Caucasian and nonconsanguineous. Patients attended UHS or samples were couriered to UHS from satellite referral centres, with no prescreening of nNO.

ital Southampton (UHS) for diagnostic testing between 2007 and 2013; 654 had adequate data and samples for inclusion. The population served by the centre is predominantly Caucasian and nonconsanguineous. Patients attended UHS or samples were couriered to UHS from satellite referral centres, with no prescreening of nNO. Diagnostic testing The pathway leading to diagnostic outcomes is summarised in figure 1. Details of the method are provided in the online supplementary material. FIGURE 1 Primary ciliary dyskinesia (PCD) diagnostic pathway for patients and samples. Diagnostic tests included nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA) and transmission electron microscopy (TEM). Not all patients underwent all tests. UHS: University Hospital Southampton. Patients and samples Patients were required to have been free of infection for ≥4 weeks. At UHS, demographic and clinical history was recorded using a standard form. At UHS nNO was measured [20] using a chemiluminescence analyser (NIOX Flex; Aerocrine, Solna, Sweden) aspirating nasal air from the nostril at 0.3 L·min−1 during a breath-hold manoeuvre. Based on experience, since 2007 we have considered an arbitrary cut-off of ≤30 nL·min−1. Following nNO measurement, a nasal brush biopsy provided epithelial cells for HSVMA, TEM and ALI culture. Satellite centres completed patient proformas and brush biopsies were couriered to UHS. Cells for HSVMA and ALI culture were transported in buffered medium within 3 h, while fixed samples for TEM were accepted with longer transportation times.

Patients and samples Patients were required to have been free of infection for ≥4 weeks. At UHS, demographic and clinical history was recorded using a standard form. At UHS nNO was measured [20] using a chemiluminescence analyser (NIOX Flex; Aerocrine, Solna, Sweden) aspirating nasal air from the nostril at 0.3 L·min−1 during a breath-hold manoeuvre. Based on experience, since 2007 we have considered an arbitrary cut-off of ≤30 nL·min−1. Following nNO measurement, a nasal brush biopsy provided epithelial cells for HSVMA, TEM and ALI culture. Satellite centres completed patient proformas and brush biopsies were couriered to UHS. Cells for HSVMA and ALI culture were transported in buffered medium within 3 h, while fixed samples for TEM were accepted with longer transportation times. Laboratory analyses HSVMA and TEM were analysed in blinded fashion by PCD-specialist microscopists (online supplementary material). At least six healthy strips of ciliated epithelium were recorded at 500 frames per second (fps). Sequences were played back at 30 fps to observe the CBP and calculate CBF. CBP was qualitatively assessed as normal, dyskinetic (static, uncoordinated, rotational, reduced beat amplitude, slow or hyperfrequent), valid-inconclusive despite adequate sample or invalid-inconclusive due to inadequate sample. HSVMA was only reported normal if both CBF (normal range 11–20 Hz) and CBP were normal.

F. CBP was qualitatively assessed as normal, dyskinetic (static, uncoordinated, rotational, reduced beat amplitude, slow or hyperfrequent), valid-inconclusive despite adequate sample or invalid-inconclusive due to inadequate sample. HSVMA was only reported normal if both CBF (normal range 11–20 Hz) and CBP were normal. TEM analysis was carried out if HSVMA was abnormal or inconclusive. ≥100 cilia were imaged in transverse section at ×60 000 magnification for the assessment of axonemal structure. Using in-house normative data, quantitative analysis determined ciliary ultrastructure as normal, abnormal, valid-inconclusive or inadequate-inconclusive. HSVMA and/or TEM were reanalysed following ALI culture or repeat biopsy unless results were normal. Diagnostic decisions Data were reviewed at multidisciplinary team meetings, attended by a clinician, an HSVMA microscopist and a TEM microscopist. All clinical and diagnostic data were considered when agreeing the diagnostic outcome as PCD-positive, PCD-negative or inconclusive.

TEM analysis was carried out if HSVMA was abnormal or inconclusive. ≥100 cilia were imaged in transverse section at ×60 000 magnification for the assessment of axonemal structure. Using in-house normative data, quantitative analysis determined ciliary ultrastructure as normal, abnormal, valid-inconclusive or inadequate-inconclusive. HSVMA and/or TEM were reanalysed following ALI culture or repeat biopsy unless results were normal. Diagnostic decisions Data were reviewed at multidisciplinary team meetings, attended by a clinician, an HSVMA microscopist and a TEM microscopist. All clinical and diagnostic data were considered when agreeing the diagnostic outcome as PCD-positive, PCD-negative or inconclusive. Positive diagnosis was reported in patients with typical clinical history, usually with at least two abnormal diagnostic tests (TEM, HSVMA and nNO), but in patients with a strong history (e.g. sibling with PCD or “full” clinical phenotype (e.g. neonatal respiratory distress at term followed by daily wet cough, persistent rhinitis and glue ear, often associated with episodes of upper and lower respiratory tract infection)), we occasionally reported a positive diagnosis based on “hallmark” TEM or repeated HSVMA consistent with PCD. CBP was considered positive if the pattern was typical of PCD rather than secondary ciliary dyskinesia, determined either from two brushing biopsies or from one brushing biopsy with reanalysis following ALI culture.

ccasionally reported a positive diagnosis based on “hallmark” TEM or repeated HSVMA consistent with PCD. CBP was considered positive if the pattern was typical of PCD rather than secondary ciliary dyskinesia, determined either from two brushing biopsies or from one brushing biopsy with reanalysis following ALI culture. Negative diagnosis was reported if 1) HSVMA with or without TEM was normal or 2) HSVMA and TEM abnormalities were consistent with secondary rather than primary dyskinesia and normal nNO (if available). A valid-inconclusive diagnosis was reported if, on repeated testing, adequate samples had subtle abnormalities not “classical” for PCD but outside the range of our experience of secondary defects. It was considered that these patients might have subtle or rare variants of ciliary phenotype. Patients were therefore told that the diagnosis was equivocal, with the recommendation that they received appropriate treatment (e.g. airway clearance or treatment of exacerbations). They were investigated for other causes of their symptoms (e.g. cystic fibrosis genotype and immunology) and were kept under review for further testing as new tests become available (e.g. new PCD-associated mutations).

ation that they received appropriate treatment (e.g. airway clearance or treatment of exacerbations). They were investigated for other causes of their symptoms (e.g. cystic fibrosis genotype and immunology) and were kept under review for further testing as new tests become available (e.g. new PCD-associated mutations). If TEM and HSVMA were inconclusive due to inadequate samples, e.g. sparse cilia, the diagnostic outcome was invalid-inconclusive and patients were invited for repeat testing. Patients with normal TEM in isolation were considered invalid-inconclusive, since TEM misses 20–30% of PCD cases [21]. Patients with nNO ≤30 nL·min−1 were deemed likely to have PCD, but it was never accepted as a lone diagnostic test. Statistical analysis Data were prospectively recorded in an Access database (Microsoft, Redmond, WA, USA) and exported to SPSS Statistics 21 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6 (La Jolla, CA, USA) for analysis. Additional details are listed in the online supplementary material. The distribution of clinical data were examined by univariate analysis. Prevalence of categorical variables was presented as percentages, and Chi-squared and Fisher's exact tests assessed proportional differences. For continuous variables mean±sd with two-tailed parametric (t) or nonparametric (×2, Mann–Whitney) tests were presented. p<0.05 was considered statistically significant.

tivityƒ 1.00 1.00 1.00 0.90 0.61 0.65 0.65 Net specificityƒ 0.87 0.92 0.87 1.00 1.00 1.00 1.00 Data are presented as n, unless otherwise stated. PPV: positive predictive value; NPV: negative predictive value. #: n=180; ¶: n=36; +: n=51; §: n=43; ƒ: the net sensitivity and specificity were calculated for combined tests. Excellent net sensitivity and specificity were achieved upon simultaneous testing of HSVMA with nNO (100% and 87%, respectively) or HSVMA with TEM (100% and 92%, respectively) or all three tests (100% and 87%, respectively). Accuracy of individual tests using HSVMA or TEM as the reference standard We calculated the accuracies of individual tests assuming HSVMA and TEM to be the reference standard for diagnosing PCD. When TEM analysis was considered as the reference standard, HSVMA sensitivity and specificity were 1.00 and 0.86, respectively, and nNO (≤30 nL·min−1) sensitivity and specificity were 0.95 and 0.89, respectively. When HSVMA was considered as the reference standard, TEM sensitivity was 0.48 (95% CI 0.38–0.59) and specificity was 1.00 (95 % CI 0.98–1.00); nNO (≤30 nL·min−1) sensitivity was 0.50 and specificity was 0.96 (online supplementary table S2). Discussion Our large cross-sectional study provides prospectively collected outcome data following a comprehensive range of PCD diagnostic tests. Our diagnostic algorithm varies from some centres [23], but the findings may contribute to the development of international consensus.

alysis. Prevalence of categorical variables was presented as percentages, and Chi-squared and Fisher's exact tests assessed proportional differences. For continuous variables mean±sd with two-tailed parametric (t) or nonparametric (×2, Mann–Whitney) tests were presented. p<0.05 was considered statistically significant. For repeated sampling, the most recent test result was used, although all data were considered by the multidisciplinary team when deciding final diagnostic outcome (figure 1). Patients with inadequate-inconclusive outcomes were excluded for analysis of test accuracy. The sensitivity and specificity of the individual tests were determined firstly based on definite positive or negative diagnostic outcome, and then assuming all valid-inconclusive outcomes to be truly positive or negative (using multidisciplinary diagnosis as a reference standard). Receiver operating characteristic (ROC) curves were constructed for nNO and CBF. Further accuracy analysis was completed using HSVMA and TEM as the reference standards. Additionally, sensitivity, specificity and predictive values were calculated for those who underwent all diagnostic tests (n=180) and compared with the whole study population (using multidisciplinary diagnosis as a reference standard); the whole population was then further stratified into: 1) the full protocol at UHS or 2) partial protocol when samples were couriered to UHS (nNO measurements not taken). We also allowed for the fact that those aged <5 years did not have nNO readings measured.

using multidisciplinary diagnosis as a reference standard); the whole population was then further stratified into: 1) the full protocol at UHS or 2) partial protocol when samples were couriered to UHS (nNO measurements not taken). We also allowed for the fact that those aged <5 years did not have nNO readings measured. For those who underwent all three tests (n=180), theoretical combination testing approaches [22] were used to determine net sensitivity and specificity of simultaneous (two or more tests in parallel; positive result if any test was abnormal) and sequential (second test only performed if first test(s) abnormal) diagnostic protocols. Net sensitivity/specificity used the addition rule of probability for simultaneous tests and the multiplication rule of probability for sequential tests.

(two or more tests in parallel; positive result if any test was abnormal) and sequential (second test only performed if first test(s) abnormal) diagnostic protocols. Net sensitivity/specificity used the addition rule of probability for simultaneous tests and the multiplication rule of probability for sequential tests. Results Study population We assessed 868 patients between April 2007 and December 2013 (48% male; median (range) age 7 (0–79) years). 517 (60%) attended the UHS clinic in person (figure 2a) and 351 samples were delivered by courier (figure 2b). 75 (9%) patients had a positive diagnosis, 566 (65%) had a negative diagnosis and 13 (1%) had inconclusive diagnostic outcome despite adequate samples. 214 (25%) patients had invalid-inconclusive results due to inadequate data at the time of the study, of whom 113 (13%) patients had only TEM samples sent from satellite clinics. Invalid-inconclusive outcomes were excluded from the analyses, resulting in a study population of 654, of which 641 had a definitive positive or negative outcome. The characteristics of the positive, negative and inconclusive patients are shown in table 1. FIGURE 2 The diagnostic investigations and outcomes of patients seen a) at the diagnostic centre at University Hospital Southampton (UHS) or b) having had samples sent by courier to UHS from a satellite respiratory clinic. Patients were diagnosed as primary ciliary dyskinesia (PCD)-positive, PCD-negative or valid-inconclusive (VI). Invalid-inconclusive (II) results due to inadequate samples or data are shown, but were subsequently excluded from accuracy analyses. Diagnostic tests included nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA) and transmission electron microscopy (TEM).

ive, PCD-negative or valid-inconclusive (VI). Invalid-inconclusive (II) results due to inadequate samples or data are shown, but were subsequently excluded from accuracy analyses. Diagnostic tests included nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA) and transmission electron microscopy (TEM). TABLE 1 Clinical characteristics of the referral population grouped by positive, negative, valid-inconclusive and invalid-inconclusive diagnostic outcomes

ive, PCD-negative or valid-inconclusive (VI). Invalid-inconclusive (II) results due to inadequate samples or data are shown, but were subsequently excluded from accuracy analyses. Diagnostic tests included nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA) and transmission electron microscopy (TEM). TABLE 1 Clinical characteristics of the referral population grouped by positive, negative, valid-inconclusive and invalid-inconclusive diagnostic outcomes Total referrals Positive Negative Valid-inconclusive Invalid-inconclusive Yes No NA# Yes No NA# Yes No NA# Yes No NA# Yes No NA# Subjects 868 75 566 13 214 Male 362 (42) 393 (45) 11 (13) 34 (45) 36 (48) 5 (7) 249 (44) 278 (49) 39 (7) 7 (54) 6 (46) 0 (0) 72 (34) 73 (34) 69 (32) Full-term gestation 346 (40) 102 (12) 420 (48) 66 (88) 7 (9) 2 (3) 242 (43) 80 (14) 244 (43) 8 (61) 2 (15) 3 (23) 30 (14) 13 (6) 171 (80) Sibling with PCD 32 (4) 792 (91) 44 (5) 18 (24) 55 (73) 2 (3) 9 (2) 529 (93) 28 (5) 0 (0) 11 (85) 2 (15) 5 (2) 197 (92) 12 (6) Neonatal unit 138 (16) 694 (80) 35 (4) 46 (61) 25 (33) 4 (5) 77 (14) 466 (82) 23 (4) 3 (23) 9 (69) 1 (8) 12 (6) 194 (91) 8 (4) Situs abnormality 70 (8) 788 (91) 10 (1) 33 (44) 42 (56) 0 (0) 22 (5) 537 (95) 7 (0) 4 (31) 9 (69) 0 (0) 11 (5) 200 (93) 3 (1) Cardiac abnormality 20 (2) 848 (98) 0 (0) 6 (8) 69 (92) 0 (0) 10 (2) 556 (98) 0 (0) 0 (0) 13 (100) 0 (0) 4 (2) 210 (98) 0 (0) Pulmonary symptoms 710 (82) 158 (18) 0 (0) 72 (96) 3 (4) 0 (0) 488 (86) 78 (14) 0 (0) 12 (93) 1 (7) 0 (0) 138 (64) 76 (36) 0 (0) Rhinitis 477 (55) 389 (45) 2 (0) 61 (81) 14 (19) 0 (0) 325 (57) 239 (42) 2 (1) 9 (69) 4 (31) 0 (0) 82 (38) 132 (62) 0 (0) Sinusitis 192 (22) 663 (76) 13 (2) 21 (28) 53 (71) 1 (1) 138 (24) 416 (74) 12 (2) 5 (38) 8 (62) 0 (0) 28 (13) 186 (87) 0 (0) Data are presented as n or n (%). Patients with invalid-inconclusive outcomes were excluded from the study population for analyses. NA: not available; PCD: primary ciliary dyskinesia. #: for example, children did not know their fertility status; older adults did not know neonatal details; and in a minority of cases the data were simply not recorded.

). Patients with invalid-inconclusive outcomes were excluded from the study population for analyses. NA: not available; PCD: primary ciliary dyskinesia. #: for example, children did not know their fertility status; older adults did not know neonatal details; and in a minority of cases the data were simply not recorded. Accuracy of individual diagnostic tests Analysis was dependent on the quality of the sample and many patients required repeat biopsies: 17% (113 out of 654) required one repeat; 2% (11 out of 654) required two repeats; and 0.4% (three out of 654) required three repeats. Analysis of diagnostic accuracy was based on the final successful test completed.

c tests Analysis was dependent on the quality of the sample and many patients required repeat biopsies: 17% (113 out of 654) required one repeat; 2% (11 out of 654) required two repeats; and 0.4% (three out of 654) required three repeats. Analysis of diagnostic accuracy was based on the final successful test completed. Nasal nitric oxide nNO was measured in 301 (47%) patients with a positive or negative diagnosis. nNO was significantly lower in PCD-positive patients (17±20 nL·min−1, 95% CI 10–23 nL·min−1) than negative patients (172±94 nL·min−1, 95% CI 160–183 nL·min−1) (p<0.0001) (online supplementary figure S1). ROC curve analysis showed low nNO to be a strong predictor of a multidisciplinary diagnosis of PCD (area under the curve 0.97, 95% CI 0.94–1.00) (figure 3). A cut-off of 30 nL·min−1 was sensitive (0.91, 95% CI 0.76–0.98) and specific (0.96, 95% CI 0.93–0.98) (table 2). Inclusion of eight valid-inconclusive results as PCD-positive (109.7±119 nL·min−1, 95% CI 10–209 nL·min−1) reduced sensitivity to 0.78 (95% CI 0.62–0.89) (online supplementary table S1). FIGURE 3 Receiver operating characteristic (ROC) curve analysis for ciliary beat frequency (CBF) and nasal nitric oxide (nNO) for predicting a diagnosis of primary ciliary dyskinesia (PCD) (using multidisciplinary diagnosis as the reference standard). ROC curve analysis showed that nNO ≤30 nL·min−1 (area under the curve (AUC) 0.97, 95% CI 0.94–1.00) was superior to CBF (AUC 0.92, 95% CI 0.79–1.00) as predictors of a PCD-positive diagnosis.

O) for predicting a diagnosis of primary ciliary dyskinesia (PCD) (using multidisciplinary diagnosis as the reference standard). ROC curve analysis showed that nNO ≤30 nL·min−1 (area under the curve (AUC) 0.97, 95% CI 0.94–1.00) was superior to CBF (AUC 0.92, 95% CI 0.79–1.00) as predictors of a PCD-positive diagnosis. TABLE 2 The diagnostic accuracy of nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA) and transmission electron microscopy (TEM) analysis to diagnose primary ciliary dyskinesia nNO ≤30 nL·min−1 HSVMA TEM Subjects# 301 (47) 625 (98) 368 (57) Positive patients¶ 34 (45) 60 (80) 71 (95) Negative patients+ 267 (47) 565 (100) 297 (52) True positive 31 60 56 True negative 257 526 297 False positive 10 39 0 False negative 3 0 15 Sensitivity (95% CI) 0.91 (0.76–0.98) 1.00 (0.94–1.00) 0.79 (0.68–0.88) Specificity (95% CI) 0.96 (0.93–0.98) 0.93 (0.91–0.95) 1.00 (0.99–1.00) PPV (95% CI) 0.76 (0.60–0.88) 0.61 (0.50–0.70) 1.00 (0.94–1.00) NPV (95% CI) 0.99 (0.97–1.00) 1.00 (0.99–1.00) 0.95 (0.92–0.97) Data are presented as n (%) or n, unless otherwise stated. Data were analysed for patients with conclusive positive or negative results who underwent the individual tests. PPV: positive predictive value; NPV: negative predictive value. #: n=641; ¶: n=75; +: n=566.

CI) 0.99 (0.97–1.00) 1.00 (0.99–1.00) 0.95 (0.92–0.97) Data are presented as n (%) or n, unless otherwise stated. Data were analysed for patients with conclusive positive or negative results who underwent the individual tests. PPV: positive predictive value; NPV: negative predictive value. #: n=641; ¶: n=75; +: n=566. High-speed video microscopy analysis HSVMA was performed in 625 (98%) patients including 60 PCD-positive and 565 PCD-negative cases. HSVMA was abnormal in all 60 positive patients tested. Of 565 PCD-negative patients, 39 had abnormal or equivocal HSVMA results (17 had abnormal CBF and 22 had abnormalities of CBP). HSVMA had excellent sensitivity (1.00, 95% CI 0.94–1.00) and specificity (0.93, 95% CI 0.91–0.95) (table 2). Since our definition of PCD includes abnormal ciliary function (pattern or frequency), sensitivity would be expected to approach 1.00. Inclusion of valid-inconclusive results as PCD-positive kept sensitivity high at 0.97 (95% CI 0.90–1.00) (online supplementary table S1). Subgroup analysis (e.g. UHS versus courier-delivered or age <5 years) made little difference to the sensitivity, specificity or predictive values (tables 2 and 3). TABLE 3 The diagnostic accuracy of nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA) and transmission electron microscopy (TEM) analysis to diagnose primary ciliary dyskinesia

er-delivered or age <5 years) made little difference to the sensitivity, specificity or predictive values (tables 2 and 3). TABLE 3 The diagnostic accuracy of nasal nitric oxide (nNO), high-speed video microscopy analysis (HSVMA) and transmission electron microscopy (TEM) analysis to diagnose primary ciliary dyskinesia UHS Courier <5 years >5 years <5 years >5 years HSVMA TEM nNO ≤30 nL·min−1 HSVMA TEM HSVMA TEM HSVMA TEM Subjects 126 355 80 80 Total 125 (99) 67 (53) 301 (85) 353 (99) 212 (60) 71 (89) 48 (60) 76 (95) 41 (51) Positive 8 (6) 7 (10) 34 (11) 34 (10) 33 (16) 8 (11) 17 (35) 10 (13) 14 (34) Negative 117 (94) 60 (90) 267 (89) 319 (90) 179 (84) 63 (89) 31 (65) 66 (87) 27 (66) True positive 8 6 31 34 22 8 15 10 13 True negative 111 60 257 297 179 59 31 59 27 False positive 6 0 10 22 0 4 0 7 0 False negative 0 1 3 0 11 0 2 0 1 Sensitivity (95% CI) 1.00 (0.63–1.00) 0.86 (0.42–0.98) 0.91 (0.76–0.98) 1.00 (0.90–1.00) 0.67 (0.48–0.82) 1.00 (0.63–1.00) 0.88 (0.64–0.98) 1.00 (0.69–1.00) 0.93 (0.66–0.99) Specificity (95% CI) 0.95 (0.89–0.98) 1.00 (0.94–1.00) 0.96 (0.93–0.98) 0.93 (0.90–0.96) 1.00 (0.98–1.00) 0.94 (0.85–0.98) 1.00 (0.89–1.00) 0.89 (0.79–0.96) 1.00 (0.87–1.00) PPV (95% CI) 0.57 (0.29–0.82) 1.00 (0.54–1.00) 0.76 (0.60–0.88) 0.61 (0.47–0.74) 1.00 (0.84–1.00) 0.67 (0.35–0.90) 1.00 (0.78–1.00) 0.59 (0.33–0.81) 1.00 (0.75–1.00) NPV (95% CI) 1.00 (0.97–1.00) 0.98 (0.91–1.00) 0.99 (0.97–1.00) 1.00 (0.99–1.00) 0.94 (0.90–0.97) 1.00 (0.94–1.00) 0.94 (0.80–0.99) 1.00 (0.94–1.00) 0.96 (0.82–0.99) Data are presented as n or n (%), unless otherwise stated. Analyses were stratified by patients seen at University Hospital Southampton (UHS) and samples sent by courier to UHS; then stratified further by age <5 years and ≥5 years at time of assessment. PPV: positive predictive value; NPV: negative predictive value.

9) Data are presented as n or n (%), unless otherwise stated. Analyses were stratified by patients seen at University Hospital Southampton (UHS) and samples sent by courier to UHS; then stratified further by age <5 years and ≥5 years at time of assessment. PPV: positive predictive value; NPV: negative predictive value. The mean CBF for PCD-positive patients (2.3±5.2 Hz, 95% CI 0.4–4.3 Hz) was significantly lower than for PCD-negative patients (15.4±2.3 Hz, 95% CI 15.2–15.6 Hz) (p<0.0001) (online supplementary figure S2). ROC curve analysis showed CBF to discriminate well between PCD-positive and -negative patients (AUC 0.92, 95% CI 0.79–1.00) (figure 3). However, it was not possible to derive a reliable CBF for 31 (41%) PCD-positive patients with variable CBP. Transmission electron microscopy TEM was performed on samples from 368 (57%) patients including 72 PCD-positive and 297 PCD-negative cases. 57 (79%) out of 72 PCD-positive patients had hallmark ultrastructural defects of PCD: 31% with outer dynein arm (ODA) and inner dynein arm (IDA) defects; 26% with an ODA defect; 10% with an ODA defect and suspected IDA defect; 7% with microtubule disarrangement and IDA defect; 4% with an intermittent central pair microtubule defect; and 1% with a microtubule transposition defect. 21% had “normal” ciliary ultrastructure. None of the 297 PCD-negative patients had ultrastructural changes suggestive of PCD, but secondary changes (e.g. swollen membranes or compound cilia) were fairly frequent.

t; 4% with an intermittent central pair microtubule defect; and 1% with a microtubule transposition defect. 21% had “normal” ciliary ultrastructure. None of the 297 PCD-negative patients had ultrastructural changes suggestive of PCD, but secondary changes (e.g. swollen membranes or compound cilia) were fairly frequent. TEM sensitivity was 0.79 (95% CI 0.68–0.88) and specificity was 1.0 (95% CI 0.99–1.00) (table 2). Again, subgroup analysis made little difference to the sensitivity, specificity or predictive values (tables 2 and 3). Air–liquid interface culture ALI culture was performed on 808 samples and 241 (30%) ciliated. Ciliary function was reanalysed following ALI culture in 152 (24%) patients. ALI samples confirmed a persistent abnormality of CBP in 21 out of 21 PCD-positive patients. Out of 124 PCD-negative patients, 123 had a normal CBP following ALI culture, and one patient had uncoordinated cilia, perhaps due to variable cell health.

ry function was reanalysed following ALI culture in 152 (24%) patients. ALI samples confirmed a persistent abnormality of CBP in 21 out of 21 PCD-positive patients. Out of 124 PCD-negative patients, 123 had a normal CBP following ALI culture, and one patient had uncoordinated cilia, perhaps due to variable cell health. Accuracy of combinations of tests Various combinations of diagnostic tests are undertaken at our centre (figure 2) while alternative combinations are used in other centres [23]. We calculated accuracy for the combinations of tests in 180 patients who had undergone all diagnostic tests, allowing us to consider theoretical scenarios (table 4). If nNO had been used as a screening test followed sequentially by TEM, 36 patients would have proceeded to TEM, but three out of 31 PCD patients would have been “missed” by not proceeding to further testing due to false negative nNO results; TEM would have subsequently failed to identify nine PCD patients. The net specificity for this combination of tests was excellent (100%), but net sensitivity was poor, failing to identify PCD in 12 (39%) out of 31 patients. Alternatively, if nNO had been used as a screening test followed sequentially by HSVMA, three out of 31 PCD patients would not have proceeded to HSVMA; however, HSVMA would have subsequently identified all 28 positive patients. Therefore, the net sensitivity and specificity were 90% and 100%, respectively. TABLE 4 Sensitivity and specificity of high-speed video microscopy analysis (HSVMA), ciliary beat pattern, nasal nitric oxide (nNO) and transmission electron microscopy (TEM) applied as single or combined tests, using simultaneous or sequential testing

he net sensitivity and specificity were 90% and 100%, respectively. TABLE 4 Sensitivity and specificity of high-speed video microscopy analysis (HSVMA), ciliary beat pattern, nasal nitric oxide (nNO) and transmission electron microscopy (TEM) applied as single or combined tests, using simultaneous or sequential testing Single testing Simultaneous testing Sequential two-stage testing nNO HSVMA TEM nNO + HSVMA HSVMA + TEM nNO + HSVMA + TEM 1. nNO# 2. HSVMA¶ 1. nNO# 2. TEM¶ 1. nNO + HSVMA# 2. TEM+ 1. HSVMA# 2. TEM§ Subjects 180 180 180 180 180 180 36 36 51 43 Positive 31 31 31 31 31 31 28 28 31 31 Negative 149 149 149 149 149 149 8 8 20 12 True positive 28 31 20 31 31 31 28 19 20 20 True negative 141 137 149 129 137 129 8 8 20 12 False positive 8 12 0 20 12 20 0 0 0 0 False negative 3 0 11 0 0 0 0 9 11 11 Sensitivity (95% CI) 0.90 (0.74–0.98) 1.00 (0.89–1.00) 0.65 (0.45–0.81) Specificity (95% CI) 0.95 (0.90–0.98) 0.92 (0.86–0.96) 1.00 (0.98–1.00) PPV (95% CI) 0.78 (0.61–0.90) 0.72 (0.56–0.85) 1.00 (0.83–1.00) NPV (95% CI) 0.98 (0.94–1.00) 1.00 (0.97–1.00) 0.93 (0.88–0.97) Net sensitivityƒ 1.00 1.00 1.00 0.90 0.61 0.65 0.65 Net specificityƒ 0.87 0.92 0.87 1.00 1.00 1.00 1.00 Data are presented as n, unless otherwise stated. PPV: positive predictive value; NPV: negative predictive value. #: n=180; ¶: n=36; +: n=51; §: n=43; ƒ: the net sensitivity and specificity were calculated for combined tests.

Accuracy of individual tests using HSVMA or TEM as the reference standard We calculated the accuracies of individual tests assuming HSVMA and TEM to be the reference standard for diagnosing PCD. When TEM analysis was considered as the reference standard, HSVMA sensitivity and specificity were 1.00 and 0.86, respectively, and nNO (≤30 nL·min−1) sensitivity and specificity were 0.95 and 0.89, respectively. When HSVMA was considered as the reference standard, TEM sensitivity was 0.48 (95% CI 0.38–0.59) and specificity was 1.00 (95 % CI 0.98–1.00); nNO (≤30 nL·min−1) sensitivity was 0.50 and specificity was 0.96 (online supplementary table S2). Discussion Our large cross-sectional study provides prospectively collected outcome data following a comprehensive range of PCD diagnostic tests. Our diagnostic algorithm varies from some centres [23], but the findings may contribute to the development of international consensus. A strength of this study was analyses of consecutive referrals within a national diagnostic programme, as this is likely to yield the most valid estimates of diagnostic accuracy. Although not all patients underwent all tests, this pragmatic study reflects the real patient journey. The major limitation is the lack of a “gold reference standard”; we therefore used a surrogate standard of expert multidisciplinary consensus. Since each test contributes to the final decision, sensitivity and specificity might be overestimated. Additionally, genetic testing does not currently form part of our diagnostic pathway and this is a rapidly expanding area that is used for diagnosis in many countries.

e standard of expert multidisciplinary consensus. Since each test contributes to the final decision, sensitivity and specificity might be overestimated. Additionally, genetic testing does not currently form part of our diagnostic pathway and this is a rapidly expanding area that is used for diagnosis in many countries. 11.5% of patients with adequate samples were diagnosed as PCD-positive, which is slightly lower than some centres [6, 8, 24], but is similar to or higher than others (Switzerland, Amsterdam and London (Claudia Kuehni, Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland; Eric Haarman, VU University Medical Center, Amsterdam, the Netherlands; and Claire Hogg, Royal Brompton & Harefield NHS Foundation Trust, London UK; personal communications). Higher positive rates may be seen at centres with nNO prescreening, in consanguineous populations [25] or if access to testing is restricted to those who are extremely likely to have PCD. The prevalence in referral populations will not affect the sensitivity or specificity but will alter the positive and negative predictive values of the tests.

be seen at centres with nNO prescreening, in consanguineous populations [25] or if access to testing is restricted to those who are extremely likely to have PCD. The prevalence in referral populations will not affect the sensitivity or specificity but will alter the positive and negative predictive values of the tests. High-speed video microscopy analysis HSVMA was sensitive and specific for diagnosing PCD; however, if used as a reference standard, this would lead to a high number of false positive results. In line with many European centres we consider HSVMA a first-line test, which might inflate the sensitivity; this needs further investigation in blinded studies. Since HSVMA is a qualitative test with potential subjectivity, results are regularly validated by external experts, but this does not exclude the possibility of some false negative findings. PCD-negative patients all had predominantly normal HSVMA, but often included a proportion of dyskinetic cilia probably due to recent infection or damage during sampling [26]. Some PCD patients had areas of apparently normal ciliary function, highlighting that PCD scientists require significant experience of the qualitative and quantitative range of PCD and non-PCD samples.

A, but often included a proportion of dyskinetic cilia probably due to recent infection or damage during sampling [26]. Some PCD patients had areas of apparently normal ciliary function, highlighting that PCD scientists require significant experience of the qualitative and quantitative range of PCD and non-PCD samples. HSVMA standardisation is challenging, and robust training, data validation, external audit and continued learning is in place. Our data cannot be generalised to centres where different standards apply. HSVMA requires expensive high-speed video equipment, high optical magnification and digital resolution for accurate CBP analysis, without which errors are likely. CBF is pH and temperature dependent, and we conduct analyses using pH-stable medium equilibrated to 37°C [27, 28].

o centres where different standards apply. HSVMA requires expensive high-speed video equipment, high optical magnification and digital resolution for accurate CBP analysis, without which errors are likely. CBF is pH and temperature dependent, and we conduct analyses using pH-stable medium equilibrated to 37°C [27, 28]. Inadequate samples and inconclusive results were a common issue for HSVMA. Recent reports of PCD-causing genes associated with subtle changes at HSVMA and TEM [29, 30] have confirmed our suspicion that some inconclusive results might represent disease. Moreover, samples providing inadequate cilia may be caused by mutations causing a syndrome similar to PCD associated with sparse but normal cilia [31, 32]. It is possible that some patients excluded from analyses due to inadequate samples will fall into this category. However, in our experience, inadequate samples are commonly adequate upon repeat brushing following antibiotics and when patients are well. We always recommend repeat testing for inadequate samples; some invalid-inconclusive cases did not return for testing because symptoms had resolved or an alternative diagnosis was identified.

our experience, inadequate samples are commonly adequate upon repeat brushing following antibiotics and when patients are well. We always recommend repeat testing for inadequate samples; some invalid-inconclusive cases did not return for testing because symptoms had resolved or an alternative diagnosis was identified. Transmission electron microscopy Approximately a fifth of PCD patients had apparently normal ciliary ultrastructure, confirming that TEM is unreliable in isolation [21, 33]. However, it is a vital part of the diagnostic portfolio, supporting HSVMA findings and providing a diagnosis when HSVMA is not available or inconclusive. Analysis of ciliary ultrastructure requires expensive equipment and electron microscopists experienced in the range of normality and abnormality. Some abnormalities are straightforward (e.g. ODA defect), but we have diagnosed several patients with subtle abnormalities of microtubules, supported by nNO and HSVMA, that would only be detected by an experienced microscopist analysing sufficient numbers of cilia in transverse and longitudinal section.

nd abnormality. Some abnormalities are straightforward (e.g. ODA defect), but we have diagnosed several patients with subtle abnormalities of microtubules, supported by nNO and HSVMA, that would only be detected by an experienced microscopist analysing sufficient numbers of cilia in transverse and longitudinal section. Nasal nitric oxide nNO is a recommended screening test for symptomatic patients [5, 6, 34]. At the outset of the prospective data collection in 2007, a cut-off of 30 nL·min−1 was arbitrarily set based on prior experience. Recent evidence suggests that higher cut-offs may be more useful [4, 34], and the accuracy of nNO cut-offs for screening/diagnostics needs to be standardised based on emerging evidence. 30 nL·min−1 was used clinically throughout data collection and so it is on this basis that we have analysed the data. The sensitivity and specificity of this cut-off were 0.91 and 0.96, respectively. Therefore, 9% of cases might be missed if further testing was excluded on the basis of this test in isolation. 77 nL·min−1 has recently been recommended as a cut-off [4]; this cut-off improved sensitivity in our population to 96%, but reduced specificity to 83%. In Leigh et al.’s [4] study, sensitivity to detect patients with PCD diagnosed by TEM or genetics was 0.98 while specificity was >0.75, similar to our findings. In our centre we are confident to use a cut-off of 30 nL·mL−1 because it is always alongside a HSVMA result. In our opinion, if nNO is used by referral centres to decide who to refer for testing, the higher cut-off with greater sensitivity should be used, but it is notable that 4% of cases might still be missed. We use nNO to support a positive diagnosis in patients with consistent subtle abnormalities of CBP who might otherwise be labelled inconclusive. We would be cautious to exclude a diagnosis of PCD in patients with nNO ≤30 nL·min−1, and these patients are more likely to be considered inconclusive and therefore undergo repeated testing. Only 47% of the study population underwent nNO testing, because it is not available at satellite centres and the breath-hold manoeuvre is usually technically acceptable only in those aged >5 years. However, the present article reports nNO data from 301 participants, which constitutes the largest study to date in a PCD diagnostic clinic population.

ion underwent nNO testing, because it is not available at satellite centres and the breath-hold manoeuvre is usually technically acceptable only in those aged >5 years. However, the present article reports nNO data from 301 participants, which constitutes the largest study to date in a PCD diagnostic clinic population. Accuracy of combinations of tests Data from patients who had undergone all tests (n=180) were used to calculate the accuracy for possible combinations of tests. Two-stage testing based on nNO prescreening followed by TEM potentially missed ∼40% of PCD cases, because both tests were required to be positive for a positive diagnosis [22]. However, our 30 nL·min−1 cut-off is probably too low for use as a screening threshold [34] and the previously discussed subjectivity of HSVMA needs to be taken into account. Simultaneous testing requires one positive test result for a positive diagnosis and, conversely, all tests to be negative for a negative outcome [22]. Using all three tests simultaneously (where any abnormal test leads to a positive result) sensitivity was 100%, but specificity reduced to 87%, compared to our multidisciplinary approach where no test was considered in isolation (100% sensitivity and 92% specificity).

l tests to be negative for a negative outcome [22]. Using all three tests simultaneously (where any abnormal test leads to a positive result) sensitivity was 100%, but specificity reduced to 87%, compared to our multidisciplinary approach where no test was considered in isolation (100% sensitivity and 92% specificity). Concluding comments Advances in understanding the molecular genetic basis of PCD have been made in recent years, to the extent that genetic testing is now able to detect ∼65% of PCD cases. However, genetic testing for PCD is not yet available in the UK except as a research tool [1]. As more genes are identified, genetic testing by multigene panel [1] will make genotyping a cost-effective approach. Characterisation of ciliary structure and function will continue to have a place within diagnostic processes, similar to the need for functional tests to confirm the diagnosis of cystic fibrosis [35]. Moreover, a thorough definition of disease phenotype by cilia ultrastructure, cilia beat pattern and nNO production rate will be extremely helpful in guiding genetic analyses in this genetically heterogeneous disease. The English public healthcare system does not fund immunofluorescence staining of ciliary proteins as a diagnostic test. This method is currently only able to detect abnormalities that are evident by TEM, and would therefore not improve our diagnostic accuracy. However, we anticipate that as more antibodies become available, immunofluorescence staining will prove a time- and cost-efficient diagnostic test.

proteins as a diagnostic test. This method is currently only able to detect abnormalities that are evident by TEM, and would therefore not improve our diagnostic accuracy. However, we anticipate that as more antibodies become available, immunofluorescence staining will prove a time- and cost-efficient diagnostic test. There is no single diagnostic test that can be used universally to diagnose PCD. Recent reports of PCD-causing genes (RSPH1) associated with subtle HSVMA and TEM abnormalities with normal nNO demonstrate the skill and expert microscopists needed for accurate diagnoses [29, 30, 36]. Importantly, the conduct and reporting of tests used to diagnose PCD are not standardised. We believe that the time is right to develop consensus standards for equipment, staff experience and protocols. Acknowledgements Thanks to Gary Connett, Julian Legg, Kerry Gove and Karen Lock (University Hospital Southampton NHS Foundation Trust, Southampton, UK) for their contribution to clinical assessments. Editorial comment in: Eur Respir J 2016; 47: 699–701 [DOI: 10.1183/13993003.01914-2015]. This article has supplementary material available from erj.ersjournals.com

Acknowledgements Thanks to Gary Connett, Julian Legg, Kerry Gove and Karen Lock (University Hospital Southampton NHS Foundation Trust, Southampton, UK) for their contribution to clinical assessments. Editorial comment in: Eur Respir J 2016; 47: 699–701 [DOI: 10.1183/13993003.01914-2015]. This article has supplementary material available from erj.ersjournals.com Support statement: The National Primary Ciliary Dyskinesia (PCD) Diagnostic Service at University Hospital Southampton is commissioned and funded by NHS England. Research at Southampton PCD Centre receives funding from EU-FP7 BESTCILIA 305404. PCD research in Southampton is supported by the National Institute for Health Research (NIHR) Southampton Respiratory Biomedical Research Unit and the NIHR Wellcome Trust Clinical Research Facility. J.S. Lucas, C. Kuehni, W.T. Walker and L. Behan are members of the European Respiratory Society Task Force to develop a practice guideline for diagnosis of PCD (ERS TF-2014-04). Authors are participants in COST Action BEAT-PCD (BM1407). Funding information for this article has been deposited with FundRef. Conflict of interest: Disclosures can be found alongside the online version of this article at erj.ersjournals.com

Introduction Primary ciliary dyskinesia (PCD) is a rare heterogeneous disease; genetic mutations cause functional and/or structural defects of cilia [1, 2]. This results in chronic upper and lower respiratory disease, such as progressive chronic suppurative lung disease, chronic serous otitis media (glue ear) and chronic rhinosinusitis [3]. Nearly half of PCD patients have situs inversus [4] and some have other heterotaxic syndromes or congenital heart defects [5, 6]. PCD affects about 1 in 10 000 people [2, 7]. As in most orphan diseases, research has focused on identifying the responsible genes, describing pathophysiological mechanisms and improving diagnostic tests [8–11]. Clinical characteristics have been described mainly in small case series and literature reviews. A recent meta-analysis found a wide range in reported prevalence of clinical manifestations of PCD [12]. Earlier reports on PCD in children suggested a relatively benign long-term course. This has been questioned by recent publications, which demonstrated that many adult patients develop severe lung disease with chronic Pseudomonas infection, become oxygen dependent and eventually require lung transplantation [3, 13–16]. Although growth and lung function are important predictors of severity and prognosis in many lung diseases [17], data on PCD is scant, showing contradictory results [13, 15, 18–23]. There are no age-standardised data on mortality and little is known about factors influencing long-term prognosis. For instance, it is not clear how mortality and lung function are influenced by different ultrastructural and genetic defects.

ses [17], data on PCD is scant, showing contradictory results [13, 15, 18–23]. There are no age-standardised data on mortality and little is known about factors influencing long-term prognosis. For instance, it is not clear how mortality and lung function are influenced by different ultrastructural and genetic defects. Although 7% of the population suffers from one of about 7000 rare diseases [24], research is scarce for these disorders. Research on PCD and many other rare diseases cannot utilise routine data such as mortality and hospital episode statistics because most rare diseases do not have a dedicated International Classification of Diseases revision 10 code and can therefore not be identified in routine statistics. The low numbers of patients in each individual centre call for collaborative research and international studies are essential.

spital episode statistics because most rare diseases do not have a dedicated International Classification of Diseases revision 10 code and can therefore not be identified in routine statistics. The low numbers of patients in each individual centre call for collaborative research and international studies are essential. Aware of these issues, clinicians and scientists with a strong interest in PCD formed an international focus group in 2006 and took a number of initiatives to advance PCD research. The first Task Force on PCD (2006–2009) of the European Respiratory Society (ERS) included 26 countries [1, 7, 25]. This European network, under the framework of the European Union's Seventh Framework Programme (EU FP7) project BESTCILIA (Better Experimental Screening and Treatment for Primary Ciliary Dyskinesia), joined forces with the North American Genetic Disorders of Mucociliary clearance Consortium. Two work packages of BESTCILIA aimed to improve the availability of international datasets for PCD patients: 1) by setting up a prospective international PCD registry [26], to allow standardised data collection in the future, and 2) by identifying and combining available data on PCD in a retrospective international cohort study. This article describes the aims and methods of the international PCD cohort (iPCD Cohort) and outlines how the data can be accessed for future research.

try [26], to allow standardised data collection in the future, and 2) by identifying and combining available data on PCD in a retrospective international cohort study. This article describes the aims and methods of the international PCD cohort (iPCD Cohort) and outlines how the data can be accessed for future research. Aims of the iPCD Cohort The iPCD Cohort assembles available datasets with clinical and diagnostic data from PCD patients worldwide to answer pertinent questions on clinical phenotype, disease severity, prognosis and effect of treatments in patients with this rare multiorgan disease. This combined international dataset allows investigation of PCD epidemiology in a large international study population in order to: 1) describe the spectrum of clinical phenotypes and disease severity in PCD patients by age, sex and time period of diagnosis; 2) describe short-term and long-term prognosis of PCD, looking at important outcomes such as growth, lung function and respiratory failure, bacterial colonisation, hearing loss, fertility, and mortality; and 3) identify predictors of long-term outcomes such as age at diagnosis, clinical phenotype, ultrastructural defects, genotype and clinical care. Methods Study design The iPCD Cohort is a retrospective international cohort, combining available data on PCD from national or local registries and clinical or diagnostic databases. All participating centres delivered retrospectively collected data; new centres joining the iPCD Cohort in the future can also participate with retrospectively collected data.

is a retrospective international cohort, combining available data on PCD from national or local registries and clinical or diagnostic databases. All participating centres delivered retrospectively collected data; new centres joining the iPCD Cohort in the future can also participate with retrospectively collected data. Identification of eligible datasets Systematic literature search In order to identify published data and eligible datasets we performed a systematic literature search for studies describing clinical manifestations in patients with PCD [12]. We searched the online databases PubMed, Embase and Scopus for studies published since 1980, without restrictions on language or study design, including studies containing information on clinical manifestations of 10 or more PCD patients. We also checked the reference lists of articles to find additional studies. We identified 52 different studies and invited all corresponding authors to participate in the iPCD Cohort. Other sources Using the database set up in a previous European PCD survey [1, 5, 7] and personal contacts from the ERS PCD Task Force, we contacted all clinicians who had reported treating PCD patients and asked them to collaborate. This included physicians from Europe, North and South America, the Middle East, and Australia.

ources Using the database set up in a previous European PCD survey [1, 5, 7] and personal contacts from the ERS PCD Task Force, we contacted all clinicians who had reported treating PCD patients and asked them to collaborate. This included physicians from Europe, North and South America, the Middle East, and Australia. Data pooling and standardisation Overall, diagnostic definitions have changed over the years. Cut-offs for diagnostic test results depend on laboratory conditions, techniques and equipment used (e.g. the reference range for ciliary beat frequency is dependent on the temperature at which analyses are made). Definitions of many clinical manifestations also vary between countries and centres. We observed considerable heterogeneity in content and format between the delivered datasets. For this reason, and to obtain comparable data for pooled analyses, we developed a standardised dataset. We discussed the content and format of this dataset with all collaborators, who include experts on PCD diagnostics and clinical care, and with PCD patient representatives, participating at BESTCILIA meetings. For diagnostic tests results, in particular, we chose the use of categorical variables in order to achieve standardisation and avoid misclassification at the time of analysis. Content and format of the variables were tailored to the availability of retrospective data, as identified by examination of the received datasets and the prospective international PCD registry [26]. This facilitates the close collaboration between the two datasets for future research.

fication at the time of analysis. Content and format of the variables were tailored to the availability of retrospective data, as identified by examination of the received datasets and the prospective international PCD registry [26]. This facilitates the close collaboration between the two datasets for future research. Diagnostic subgroups PCD diagnostics have evolved rapidly in recent years [27]. Initially, diagnosis was based on the Kartagener symptoms triad [28] and on transmission electron microscopy (TEM) findings. Then light microscopy and later high-frequency video microscopy (VM) were introduced in the diagnostic algorithm. Currently, recommendations include a combination of TEM, VM, nasal nitric oxide (nNO) and genetic testing [11], but availability of tests differs between countries [27]. Therefore, not all PCD patients have been diagnosed according to current recommendations. The iPCD Cohort includes patients diagnosed since 1964, when diagnostic criteria were different. As a result of this, we defined three different diagnostic subgroups based on the results of the available tests. We used the recent guidelines of the ERS PCD Diagnostics Task Force [11] to define “definite PCD” as hallmark TEM findings and/or bi-allelic PCD genetic mutation. “Probable PCD” was defined when patients had abnormal VM findings and/or low nNO, using a cut-off of 77 nL·min−1, as previously published [29]. All patients who had negative or ambiguous test results, or had not been tested so far, were defined as having “clinical PCD diagnosis”. These patients are followed up and treated as PCD at the collaborating centres based on a combination of several of the following features: situs anomalies, persistent cough, persistent rhinitis, chronic or recurrent upper or lower respiratory infections and history of neonatal respiratory symptoms in term infants, based on consensus statements and guidelines available to age [1, 11].

ing centres based on a combination of several of the following features: situs anomalies, persistent cough, persistent rhinitis, chronic or recurrent upper or lower respiratory infections and history of neonatal respiratory symptoms in term infants, based on consensus statements and guidelines available to age [1, 11]. What information is collected The iPCD Cohort includes retrospectively collected patient data on the following 11 thematic categories (table 1): 1) general information, 2) results of diagnostic tests, 3) baseline characteristics, 4) growth and lung function, 5) clinical manifestations, 6) therapy, 7) microbiology, 8) imaging, 9) surgical interventions, 10) neonatal period, and 11) family history. Details on all variables included in the standardised dataset and information on the coding of variables are included in online supplementary table S1. TABLE 1 Description of data collected in the international primary ciliary dyskinesia cohort (iPCD Cohort) and centres contributing to the different modules

ly history. Details on all variables included in the standardised dataset and information on the coding of variables are included in online supplementary table S1. TABLE 1 Description of data collected in the international primary ciliary dyskinesia cohort (iPCD Cohort) and centres contributing to the different modules Type of data Collected variables Contributing centres# General information¶ Patient and centre identifiers, and demographic data (e.g. date of birth, sex, ethnicity, date of last follow-up, date last known alive) All centres Diagnostics¶ Diagnostic status of PCD, date of diagnosis, dates, and results of diagnostics test performed (e.g. nNO testing, VM, TEM, genetic testing) All centres Baseline characteristics¶ Information on PCD defects (e.g. situs anomalies, congenital heart defects, brain cilia dysfunction, retinitis pigmentosa, renal problems, fertility problems) and relevant comorbidities unconnected with PCD All centres Lung function Lung function date, somatometric measurement, spirometric measurements and other lung function tests (e.g. multiple breath washout) All centres except those in Northern America Clinical manifestations Date of clinical visit, information on symptoms and signs (e.g. cough, upper and lower respiratory infections, hearing impairment), exercise limitation and active or passive smoking within the last 3 months AR, AU, CH, CY, DE2, DE3, DK, FR, IL, IT, NL, NO, PL, RS, TR, UK2, UK3 Therapy Date of prescription, information on inhaled and nasal medication (e.g. bronchodilators, inhaled corticosteroids, nasal sprays), antibiotics prescription, oxygen therapy, mechanical ventilation and physiotherapy AU, CH, CY, DE3, IL, IT, NO, PL, RS, TR, UK3 Microbiology Date of microbiology examination, type of sample and isolated bacteria AR, BE, CH, CY, DE2, DE3, DK, NO, PL, RS, TR, UK2, UK3 Imaging Date of chest imaging examination, type of chest imaging, information on findings (e.g. bronchiectases, atelectases, infiltrations) and information on other types of imaging BE, CH, CY, DE2, DE3, FR, IL, IT, NO, RS, TR, UK2, UK3 Surgical interventions Date and type of surgery AR, AU, BE, CH, CY, DE3, IT, NL, NO, RS, TR Neonatal period Symptoms and signs of neonatal period (e.g.

formation on findings (e.g. bronchiectases, atelectases, infiltrations) and information on other types of imaging BE, CH, CY, DE2, DE3, FR, IL, IT, NO, RS, TR, UK2, UK3 Surgical interventions Date and type of surgery AR, AU, BE, CH, CY, DE3, IT, NL, NO, RS, TR Neonatal period Symptoms and signs of neonatal period (e.g. neonatal respiratory distress syndrome, neonatal cough, neonatal rhinitis), need for intensive care and ventilation AU, BE, CH, CY, DE2, DE3, FR, IL, IT, NL, NO, RS, TR, UK3 Family history Number of siblings, PCD affected siblings and other family members, patient ID of PCD affected family members, and relationship of affected family members AR, AU, BE, CH, CY, DE2, IL, IT, NO, RS, TR, UK3 nNO: nasal nitric oxide; VM: light or high-frequency video microscopy; TEM: transmission electron microscopy. #: AR: Argentina; AU: Australia; BE: Belgium; CH: Switzerland; CY: Cyprus; DE1: Bochum, Germany, DE2: Muenster, Germany; DE3: Hannover, Germany; DK: Denmark; FR: France; IL: Israel; IT: Italy; NL: the Netherlands; NO: Norway; PL: Poland; RS: Serbia; TR: Turkey; UK1: Paediatric Pulmonology Dept, Brompton, UK; UK2: Adult Pulmonology Dept, Brompton, UK; UK3: Southampton, UK. ¶: mandatory data for all centres.

ermany, DE2: Muenster, Germany; DE3: Hannover, Germany; DK: Denmark; FR: France; IL: Israel; IT: Italy; NL: the Netherlands; NO: Norway; PL: Poland; RS: Serbia; TR: Turkey; UK1: Paediatric Pulmonology Dept, Brompton, UK; UK2: Adult Pulmonology Dept, Brompton, UK; UK3: Southampton, UK. ¶: mandatory data for all centres. Infrastructure for data delivery and data rights The iPCD Cohort is hosted at the Institute of Social and Preventive Medicine at the University of Bern, Switzerland. Research is performed in close collaboration with all data contributors. We have set up a safe information technology platform (Sharepoint) for uploading already available anonymised datasets. We organised telephone conferences to offer extra support during the initial steps of the data delivery, and to discuss questions and comments from the contributing partners. To further facilitate data delivery in the standardised dataset for new partners we created a web-based platform, using the software Research Electronic Data Capture (REDCap) developed at Vanderbilt University (Nashville, TN, USA), which is widely used in the academic research community [30], and allows data entry and extraction in various formats. Detailed instructions explain how data can be entered. The REDCap environment is completely secure and each contributor only has access to the data of their own centre. We collaborated closely with centres that already had national registries and large retrospective datasets, to help them recode the data into the standardised format, and we performed quality controls of the delivered data (e.g. checks for coding errors and plausibility of entered values and dates). Each partner was responsible for ensuring that the delivered data were anonymous and in accordance with the national/local data protection laws. More information on data protection can be found in the online supplementary material under Data protection/Ethics. We drafted agreements for data delivery and publication (see online supplementary material), which leave all rights with data contributors.

and in accordance with the national/local data protection laws. More information on data protection can be found in the online supplementary material under Data protection/Ethics. We drafted agreements for data delivery and publication (see online supplementary material), which leave all rights with data contributors. Data analysis For each planned analysis, all eligible data are validated, cleaned and standardised. We identify outliers and implausible values, and if necessary we contact contributors to resolve any issues encountered. Based on the research question, we use a one- or two-stage random effect individual patient data meta-analysis approach. As PCD diagnostics has evolved quickly and not all patients have diagnostic information that is up to current standards, all analyses will be stratified by level of diagnostic certainty, using the diagnostic subgroups described previously. Whenever relevant, we will perform additional sensitivity analysis including only patients with definite PCD, based on the recent guidelines of the ERS PCD Diagnostics Task Force [11], and will compare these results with the overall analysis results.

ostic certainty, using the diagnostic subgroups described previously. Whenever relevant, we will perform additional sensitivity analysis including only patients with definite PCD, based on the recent guidelines of the ERS PCD Diagnostics Task Force [11], and will compare these results with the overall analysis results. Funding The setting up of the iPCD Cohort (salaries, consumables and equipment) was funded by the EU FP7 project BESTCILIA (http://bestcilia.eu) and several Swiss funding bodies, including the Lung Leagues of Bern, St Gallen, Vaud, Ticino and Valais and the Milena Carvajal Pro-Kartagener Foundation. Data collection and management at each site was funded according to local arrangements. Most participating researchers and data contributors participate in the COST Action BEAT-PCD: Better Evidence to Advance Therapeutic options for PCD (BM 1407; www.beatpcd.org). Infrastructure is provided for free by the University of Bern, where the data are pooled and stored.

as funded according to local arrangements. Most participating researchers and data contributors participate in the COST Action BEAT-PCD: Better Evidence to Advance Therapeutic options for PCD (BM 1407; www.beatpcd.org). Infrastructure is provided for free by the University of Bern, where the data are pooled and stored. Results Current dataset The iPCD Cohort is an ongoing effort where new centres can still join and participating centres can add data for new patients. As of April 2016, data from 21 single centres or consortia from 18 countries had been contributed. The pooled dataset contained data on 3013 patients (figure 1). Some countries which have a national reference centre for all paediatric and adult PCD patients (Cyprus, Denmark and France) or a national PCD registry (Italy and Switzerland) contributed their national dataset. Other countries contributed data from their consortia of several centres (Israel, USA and Canada) or datasets from single-centre studies (all remaining centres). The number of patients per centre ranged from 10 to 436 (table 2). Two paediatric centres contributed datasets only with patients <18 years and one with adult patients (≥20 years old). Five paediatric centres additionally included a few young adults. The remaining 13 centres submitted datasets that included both paediatric and adult patients with an age range from 0 to 77 years. The percentage of males ranged from 38% to 68% among datasets. General information, results of diagnostic tests and baseline characteristics (Modules 1–3) are in the basic mandatory dataset, and thus available from all centres and for all 3013 patients or the majority of them (tables 1 and 3). In addition to these baseline data, several centres have also contributed data to the other modules (table 3). Data on growth are currently available for more than 1500 patients and on lung function for more than 1000 patients. Additional data, such as microbiology or perinatal history, have been delivered by several groups (table 1 and figure 1) and for several hundreds of patients (table 3). Longitudinal data have been contributed for 542 patients from 10 countries (table 2), with a follow-up period ranging from 2 to 20 years. TABLE 2 Characteristics of the international primary ciliary dyskinesia cohort (iPCD Cohort)

by several groups (table 1 and figure 1) and for several hundreds of patients (table 3). Longitudinal data have been contributed for 542 patients from 10 countries (table 2), with a follow-up period ranging from 2 to 20 years. TABLE 2 Characteristics of the international primary ciliary dyskinesia cohort (iPCD Cohort) Country Principal investigator Patients n Age range years# Males % Type of data delivered Data richness¶ Argentina S. Scigliano 101 6–57 42 Cross-sectional Intermediate Australia L. Morgan 109 0–76 60 Longitudinal High Belgium M. Boon 82 0–69 45 Cross-sectional High Cyprus P. Yiallouros 31 0–66 48 Longitudinal High Denmark K.G. Nielsen 120 0–70 48 Longitudinal Intermediate France A. Clement 337 0–69 52 Longitudinal High Germany (Bochum) C. Koerner-Rettberg 64 0–27 40 Cross-sectional High Germany (Muenster) H. Omran 436 3–75 52 Cross-sectional Basic Germany (Hannover) N. Schwerk 37 0–39 68 Longitudinal High Israel I. Amirav 210 0–60 56 Cross-sectional High Italy Italian PCD group 331 0–73 50 Cross-sectional Intermediate The Netherlands E. Haarman 82 3–69 50 Longitudinal Intermediate Norway S. Crowley 23 0–18 65 Longitudinal High Poland H. Mazurek 105 1–22 56 Cross-sectional High Serbia Z. Zivkovic 10 6–19 45 Longitudinal High Switzerland Swiss PCD group 108 3–70 51 Longitudinal High Turkey B. Karadag 37 3–21 43 Cross-sectional Intermediate UK (Paediatric Pulmonology Dept, Brompton) C. Hogg 116 1–18 47 Cross-sectional Basic UK (Adult Pulmonology Dept, Brompton) M. Loebinger 152 20–76 38 Cross-sectional Basic UK (Southampton) J. Lucas 104 0–68 49 Longitudinal Intermediate USA/Canada Genetic Diseases of Mucociliary Clearance Consortium 418 0–77 44 Cross-sectional Basic iPCD Cohort 3013 0–77 50 #: at time of data delivery; ¶: semiquantitative measure based on the number of delivered variables.

2 20–76 38 Cross-sectional Basic UK (Southampton) J. Lucas 104 0–68 49 Longitudinal Intermediate USA/Canada Genetic Diseases of Mucociliary Clearance Consortium 418 0–77 44 Cross-sectional Basic iPCD Cohort 3013 0–77 50 #: at time of data delivery; ¶: semiquantitative measure based on the number of delivered variables. TABLE 3 Available data from different modules of the standardised dataset of 3013 primary ciliary dyskinesia (PCD) patients in the international PCD cohort (iPCD Cohort) (April 2016) Type of data Patients General information 3013 (100) Diagnostics 3013 (100) nNO 1021 (33) TEM 1913 (62) VM 1088 (35) Genetics 276 (9) Baseline characteristics# 2286 (74) Growth 1609 (53) Lung function 1042 (34) Clinical manifestations 1352 (44) Therapy 843 (27) Microbiology 732 (24) Imaging 526 (17) Neonatal period 1179 (38) Data are presented as n (%). nNO: nasal nitric oxide; TEM: transmission electron microscopy; VM: light or high-frequency video microscopy. #: mainly data on situs anomalies and congenital heart disease. FIGURE 1 Countries contributing data to the international primary ciliary dyskinesia cohort (iPCD Cohort). The circle size reflects the size of the dataset and the shades of grey reflect the data richness (semiquantitative measure based on the number of delivered variables).

Type of data Patients General information 3013 (100) Diagnostics 3013 (100) nNO 1021 (33) TEM 1913 (62) VM 1088 (35) Genetics 276 (9) Baseline characteristics# 2286 (74) Growth 1609 (53) Lung function 1042 (34) Clinical manifestations 1352 (44) Therapy 843 (27) Microbiology 732 (24) Imaging 526 (17) Neonatal period 1179 (38) Data are presented as n (%). nNO: nasal nitric oxide; TEM: transmission electron microscopy; VM: light or high-frequency video microscopy. #: mainly data on situs anomalies and congenital heart disease. FIGURE 1 Countries contributing data to the international primary ciliary dyskinesia cohort (iPCD Cohort). The circle size reflects the size of the dataset and the shades of grey reflect the data richness (semiquantitative measure based on the number of delivered variables). Patient characteristics Characteristics of the 3013 patients included in the iPCD Cohort are described in table 4. 49% (1490) were male. 71% of patients were followed up in European centres (39% in Western Europe), 9% in Western Asia, 17% in America and 4% in Australia. More than half of the patients (table 4) have a definite PCD diagnosis based the recent guidelines of the ERS PCD Diagnostics Task Force [11]. 14% of patients were defined as probable PCD based on their diagnostic test results. In 30% of patients the test results were ambiguous or the diagnostic algorithm had not been concluded and the diagnosis was based on clinical grounds based on existing consensus or guidelines. Median (range) current age of patients was 18 (1–92) years (figure 2). Children aged between 10 and 19 years were the largest age group (38%), followed by younger children (0–9 years) and young adults (20–29 years) at 17% and 18%, respectively. The percentage of patients in the remaining age groups had a declining trend from 10% for patients aged 30–39 years to 4% for patients aged >60 years. TABLE 4 Basic characteristics of the 3013 primary ciliary dyskinesia (PCD) patients included in the international PCD cohort (iPCD Cohort) (April 2016)

8%, respectively. The percentage of patients in the remaining age groups had a declining trend from 10% for patients aged 30–39 years to 4% for patients aged >60 years. TABLE 4 Basic characteristics of the 3013 primary ciliary dyskinesia (PCD) patients included in the international PCD cohort (iPCD Cohort) (April 2016) Patients 3013 (100) Sex Male 1490 (49) Female 1523 (51) Location of country of residence# Australia 109 (4) Northern Europe 515 (17) Western Europe 1146 (39) Eastern Europe 105 (4) Southern Europe 341 (11) Western Asia 278 (9) Northern America 418 (14) Southern America 101 (3) Diagnostic information Definite PCD diagnosis¶ 1718 (56) Probable PCD diagnosis+ 420 (14) Clinical diagnosis only 875 (30) Age years 0–9 517 (17) 10–19 1156 (38) 20–29 553 (18) 30–39 316 (10) 40–49 205 (7) 50–59 137 (5) ≥60 129 (4) Data are presented as n (%). #: based on geographical region definitions of the United Nations Statistic Division (August 2016) (Northern Europe: Denmark, Norway, UK; Western Europe: Belgium, France, Germany, Switzerland, the Netherlands; Eastern Europe: Poland; Southern Europe: Italy, Serbia; Western Asia: Cyprus, Israel, Turkey; Northern America: Canada, USA; Southern America: Argentina); ¶: defined as hallmark PCD electron microscopy findings and/or bi-allelic gene mutation identified based on the European Respiratory Society PCD Diagnostics Task Force guidelines [11]; +: abnormal light or high-frequency video microscopy finding and/or low (≤77 nL·min−1) nasal nitric oxide value.

n America: Argentina); ¶: defined as hallmark PCD electron microscopy findings and/or bi-allelic gene mutation identified based on the European Respiratory Society PCD Diagnostics Task Force guidelines [11]; +: abnormal light or high-frequency video microscopy finding and/or low (≤77 nL·min−1) nasal nitric oxide value. FIGURE 2 Age distribution of 3013 primary ciliary dyskinesia (PCD) patients included in the international PCD cohort (iPCD Cohort), stratified by sex: a) male and b) female (April 2016).

n America: Argentina); ¶: defined as hallmark PCD electron microscopy findings and/or bi-allelic gene mutation identified based on the European Respiratory Society PCD Diagnostics Task Force guidelines [11]; +: abnormal light or high-frequency video microscopy finding and/or low (≤77 nL·min−1) nasal nitric oxide value. FIGURE 2 Age distribution of 3013 primary ciliary dyskinesia (PCD) patients included in the international PCD cohort (iPCD Cohort), stratified by sex: a) male and b) female (April 2016). Discussion The iPCD Cohort is the largest PCD dataset available to date. It includes data from 3013 PCD patients from 18 countries; more than half of them have a definite PCD diagnosis using current standards. The main strength of the iPCD Cohort is the large number of patients it includes. This makes it a valuable source for research, especially in the field of rare diseases where even large national reference centres may follow a small number of patients. In addition to the basic characteristics and diagnostic test results, which are available from all centres and for more than 3000 patients, the iPCD Cohort includes data from over 1000 patients on neonatal symptoms, growth, lung function and clinical characteristics, and from several hundred patients on other topics such as microbiology, imaging and therapy (e.g. growth, lung function, clinical manifestations) from a number of centres; these are higher numbers of patients than were ever previously included in publications on these topics. What is important is that most centres have included almost complete datasets in the different thematic modules, in cases where they have contributed data to them. Another strength is the multinational nature of the iPCD Cohort. It offers the possibility, unique for rare diseases, to assess differences in PCD characteristics between countries and ethnic groups, and variations in healthcare (diagnostics and treatment). The iPCD Cohort includes patients of all age groups and although the paediatric population is larger, this offers the opportunity to study the course of the disease through life. Preliminary analyses on growth, nutritional status and lung function from the iPCD Cohort have been presented at international conferences, illustrating the quality of the dataset and the feasibility of future studies using the iPCD Cohort. The user-friendly and safe environment of REDCap for the standardised entry of the data is an additional strength. Data contributors can easily type in their data following simple instructions and access it any time with password-protected accounts (box 1). In addition, they can easily export their dataset for their own analyses.

iendly and safe environment of REDCap for the standardised entry of the data is an additional strength. Data contributors can easily type in their data following simple instructions and access it any time with password-protected accounts (box 1). In addition, they can easily export their dataset for their own analyses. Some limitations are inherent to the retrospective nature of the iPCD Cohort. For instance, there remains some heterogeneity in definitions among centres. This is not an issue for standardised measurements, such as height, weight, spirometry and microbiology/radiology test results, but is more relevant for clinical signs. Reference ranges of diagnostic test results vary, and depend greatly on the expertise, conditions, equipment and protocol used at each centre. For standardisation with comparable values, we used categorical coding for numerical values (e.g. frequency of ciliary beating has been coded as normal/decreased/increased). All the items collected in the cohort and their coding were extensively discussed and agreed among collaborators during the development period, taking into account existing national registries and the prospective international PCD registry. This ensures that all important data resources could be pooled and analysed together in future collaborative studies. Another issue is the heterogeneity of the PCD patient population concerning the diagnostic evaluation; diagnostic results can be normal or very subtle in some patients who almost certainly have PCD. Therefore, we have grouped the patients into three subgroups based on the recent diagnostics guidelines from the ERS Task Force [11] and are planning to take the level of diagnostic certainty into account in all analyses. Participating centres will be encouraged to follow these guidelines for new patients and to re-evaluate the diagnosis of patients in cases where not all testing has been concluded. Although the iPCD Cohort is an important data source for statistically meaningful research, it is not truly representative of the PCD patient population. Countries that have not developed diagnostic facilities yet are under-represented. PCD is underdiagnosed in adults, and most centres follow mainly children and younger adults. Thus, adult patients, especially those of older age, are under-represented in the Cohort.

ot truly representative of the PCD patient population. Countries that have not developed diagnostic facilities yet are under-represented. PCD is underdiagnosed in adults, and most centres follow mainly children and younger adults. Thus, adult patients, especially those of older age, are under-represented in the Cohort. Most published studies on PCD include a small number of patients. The largest published study on PCD was in the framework of the previous ERS PCD Task Force, which included 1192 patients from 26 countries [7]. In that study the researchers collected basic data from questionnaires addressed to the clinical centres of participating countries. They found a slightly higher proportion of male patients and 85% of the patients were aged <20 years. Recently, data on 201 patients from the international PCD registry have been described [26]. The authors observed 55% males and almost 50% patients aged <18 years. 22% of the included patients have only a clinical diagnosis compared with 35% in the iPCD Cohort. As these two datasets were both built in the framework of BESTCILIA there is an overlap in the patients they include. The international PCD registry aims to collect prospective data of PCD patients in a standardised way to use for future studies, while the iPCD Cohort combines available retrospective datasets of PCD patients in pooled analyses to answer important questions on PCD in a large study population. Although well-designed prospective studies have obvious strengths, it is of great importance to use all available data resources for research in rare diseases such as PCD, where there is still little evidence and management of patients is primarily based on experience derived from other diseases, such as cystic fibrosis.

though well-designed prospective studies have obvious strengths, it is of great importance to use all available data resources for research in rare diseases such as PCD, where there is still little evidence and management of patients is primarily based on experience derived from other diseases, such as cystic fibrosis. The iPCD Cohort is a valuable resource for epidemiological studies in PCD and it can be further enriched and used in the framework of the BEAT-PCD EU COST Action. BEAT-PCD aims to set up a global, Europe-led multidisciplinary network of PCD clinicians and researchers, and the aims of the epidemiological work group include using this dataset for projects on PCD epidemiology. More centres have expressed an interest in contributing data to the cohort. Some groups had originally contributed a basic cross-sectional dataset and are now adding retrospectively collected repeated measurements (longitudinal data) or they are adding new patients to their datasets. Ongoing studies analyse growth, lung function, diagnostic tests, clinical manifestations, neonatal symptoms and the role of lobectomy in PCD, using the iPCD Cohort dataset. The iPCD Cohort also contains data on treatments, imaging and microbiology, which can be used and to identify patients for nested studies on PCD. This dataset is now available to be further exploited and offers a unique opportunity to study PCD in a large international patient-based cohort with sufficient statistical power. Results will help to improve survival, care and quality of life of PCD patients.

The iPCD Cohort is a valuable resource for epidemiological studies in PCD and it can be further enriched and used in the framework of the BEAT-PCD EU COST Action. BEAT-PCD aims to set up a global, Europe-led multidisciplinary network of PCD clinicians and researchers, and the aims of the epidemiological work group include using this dataset for projects on PCD epidemiology. More centres have expressed an interest in contributing data to the cohort. Some groups had originally contributed a basic cross-sectional dataset and are now adding retrospectively collected repeated measurements (longitudinal data) or they are adding new patients to their datasets. Ongoing studies analyse growth, lung function, diagnostic tests, clinical manifestations, neonatal symptoms and the role of lobectomy in PCD, using the iPCD Cohort dataset. The iPCD Cohort also contains data on treatments, imaging and microbiology, which can be used and to identify patients for nested studies on PCD. This dataset is now available to be further exploited and offers a unique opportunity to study PCD in a large international patient-based cohort with sufficient statistical power. Results will help to improve survival, care and quality of life of PCD patients. BOX 1 Contributing to and accessing data in the international primary ciliary dyskinesia cohort (iPCD Cohort) How to contribute data

This dataset is now available to be further exploited and offers a unique opportunity to study PCD in a large international patient-based cohort with sufficient statistical power. Results will help to improve survival, care and quality of life of PCD patients. BOX 1 Contributing to and accessing data in the international primary ciliary dyskinesia cohort (iPCD Cohort) How to contribute data Centres that wish to participate to the project and contribute data can contact the iPCD Cohort to sign a data delivery agreement. They then will receive a password to access the online software REDCap and they will be able to enter their data directly. They can also upload follow-up data or add additional patients at a later time point. In case of large standardised datasets, it is possible to upload them directly. We have developed detailed instructions and offer information technology support wherever needed How to access data Centres that have entered their data using REDCap will keep constant access to their datasets and can export them directly in various formats for local analyses. We have developed detailed extraction instructions to simplify the procedure.

Centres that wish to participate to the project and contribute data can contact the iPCD Cohort to sign a data delivery agreement. They then will receive a password to access the online software REDCap and they will be able to enter their data directly. They can also upload follow-up data or add additional patients at a later time point. In case of large standardised datasets, it is possible to upload them directly. We have developed detailed instructions and offer information technology support wherever needed How to access data Centres that have entered their data using REDCap will keep constant access to their datasets and can export them directly in various formats for local analyses. We have developed detailed extraction instructions to simplify the procedure. Researchers wanting to use the iPCD Cohort dataset can propose a topic and a concept sheet describing the planned analyses and publication. All concept sheets have to be approved by all centres contributing data to the proposed analysis under question. After the participating centres agree to contribute their data and sign a publication agreement, we will prepare a partial dataset for the proposed analysis and will work closely with the lead researchers offering methodological input and support. In case additional data is collected to complete the partial dataset for a specific project, this will be added to the iPCD Cohort to enrich it after each project. For further details, contact: pcd@ispm.unibe.ch REDCap: Research Electronic Data Capture.

Researchers wanting to use the iPCD Cohort dataset can propose a topic and a concept sheet describing the planned analyses and publication. All concept sheets have to be approved by all centres contributing data to the proposed analysis under question. After the participating centres agree to contribute their data and sign a publication agreement, we will prepare a partial dataset for the proposed analysis and will work closely with the lead researchers offering methodological input and support. In case additional data is collected to complete the partial dataset for a specific project, this will be added to the iPCD Cohort to enrich it after each project. For further details, contact: pcd@ispm.unibe.ch REDCap: Research Electronic Data Capture. Supplementary material 10.1183/13993003.01181-2016.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author. iPCD cohort methods and first results ERJ-01181-2016_Supplement1 Agreements for data delivery and publication ERJ-01181-2016_Supplement2 Disclosures 10.1183/13993003.01181-2016.Supp2M. Leigh ERJ-01181-2016_Leigh P. Yiallouros ERJ-01181-2016_Yiallouros

Supplementary material 10.1183/13993003.01181-2016.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author. iPCD cohort methods and first results ERJ-01181-2016_Supplement1 Agreements for data delivery and publication ERJ-01181-2016_Supplement2 Disclosures 10.1183/13993003.01181-2016.Supp2M. Leigh ERJ-01181-2016_Leigh P. Yiallouros ERJ-01181-2016_Yiallouros Acknowledgements We thank all the patients with PCD in the cohort and their families, and especially the PCD patient organisations for their close collaboration. We also thank all the researchers in the participating centres who were involved in data collection and data entry, and worked closely with us through the whole process of participating to the iPCD Cohort. We acknowledge Jingying Wang (Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland) for her help and technical support in building the REDCap dataset. Author contributions: C.E. Kuehni developed the concept and designed the study. C.E. Kuehni, M. Goutaki and E. Maurer identified eligible datasets, drafted agreements and prepared the standardised dataset. M. Goutaki and F.S. Halbeisen cleaned and standardised the data, and performed the statistical analyses. All other authors participated in discussions for the development of the study and contributed data. C.E. Kuehni, M. Goutaki, F.S. Halbeisen and J.S. Lucas drafted the manuscript; all authors contributed to iterations and approved the final version. C.E. Kuehni and M. Goutaki take final responsibility for the contents.

All other authors participated in discussions for the development of the study and contributed data. C.E. Kuehni, M. Goutaki, F.S. Halbeisen and J.S. Lucas drafted the manuscript; all authors contributed to iterations and approved the final version. C.E. Kuehni and M. Goutaki take final responsibility for the contents. This article has supplementary material available from erj.ersjournals.com Support statement: The development of the iPCD Cohort has been funded from the European Union's Seventh Framework Programme under EG-GA 35404 BESTCILIA: Better Experimental Screening and Treatment for Primary Ciliary Dyskinesia. Primary ciliary dyskinesia research at ISPM Bern is also funded by national funding from the Lung Leagues of Bern, St Gallen, Vaud, Ticino and Valais and the Milena Carvajal Pro-Kartagener Foundation. The researchers participate in the network of COST Action BEAT-PCD: Better Evidence to Advance Therapeutic options for PCD (BM 1407). Funding information for this article has been deposited with the Open Funder Registry. Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com