The all age asthma cohort (ALLIANCE) - from early beginnings to chronic disease: a longitudinal cohort study

The ALLIANCE infrastructure is provided by the participating sites of the German Centre for Lung Research (DZL) and associated study centres, i.e. university hospitals, academic and private research institutions in Luebeck, Grosshansdorf, Borstel, Hannover, Munich, Marburg and Cologne. Direct costs of the ALLIANCE Cohort are being paid by project grants (first funding period 2011–2015 and second funding period 2016–2020) from the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, BMBF) as part of the DZL funding. All studies were approved by the local ethics committees and are registered at clinicaltrials.gov (paediatric arm: NCT02496468; adult arm: NCT02419274).

Study participants for both arms (paediatric and adult) of ALLIANCE are recruited at the participating study sites (for details see Fig. 2). In the case of children with new-onset disease, cases were additionally recruited in private practices of registered paediatricians. For these children, disease course can be followed from the beginning on, and they are rarely seen in a clinical but much more often in a primary care setting due to their early disease state. Healthy paediatric controls (age- and sex-matched) were recruited in the same centres, via notices across campus and in private practices of registered paediatricians, as well as during clinics of separate subspecialties (e.g. paediatric growth) or if scheduled for short surgical interventions.

Adult patients with asthma are recruited from the inpatient and outpatient departments of LungenClinic Grosshansdorf and of the Medical Clinical of Research Center Borstel, and from an institutional database that is used for clinical trials (Pulmonary Research Institute at LungenClinic Grosshansdorf). Healthy adult controls were also recruited from an existing institutional database and by advertisement.

For children, the following inclusion criteria apply in addition to informed consent of either parent or caretaker and of the child if aged 8 years or older: age 6 months to 18 years, term delivery (≥ 37 weeks); active/passive understanding of German. Steroid−/leukotriene receptor antagonist (LTRA)-naivety is defined as no use of inhaled or systemic corticosteroids or LTRA for at least four months prior to inclusion. Exclusion criteria for participants of the paediatric arm of ALLIANCE are: known inborn or perinatal pulmonary disease; pulmonary malformation; oxygen therapy after birth with a duration of more than 24 h; ventilator support or mechanical ventilation after birth; diagnosis of cystic fibrosis; primary ciliary dyskinesia; heart failure diagnosed after birth affecting pulmonary circulation; major respiratory diseases such as e.g. interstitial lung disease; and any current non-atopic comorbidities. Moreover, children are excluded from study visits and biomaterial collection in the case of fever of at least 38.5 °C during the last two weeks prior to the planned visit.

Childhood cases are specified as either having doctor-diagnosed wheeze during at least 2 occasions during the last 12 months (age < 6 years) or as having doctor-diagnosed asthma (age ≥ 6 years) with diagnosis according to current guidelines including lung function [10, 11]. Healthy controls are defined as children without wheeze or asthma and otherwise applying the same in- and exclusion criteria as mentioned above. Adolescent cases turning 18 years of age will automatically enter transition into the adult arm of ALLIANCE, with identical data and biomaterial collection as prior to transition.

The following inclusion criteria apply in addition to informed consent for participants who are newly recruited during adulthood: age ≥ 18 years, active/passive understanding of German, and an established diagnosis of asthma according to current guidelines [10, 11]. Participants are allowed to be current or former smokers to avoid significant selection bias. Patients with asthma and a relevant smoking history are accurately screened for features distinguishing asthma from chronic obstructive pulmonary disease (COPD): age at onset, pattern and time course of symptoms, personal and family history, variable or persistent airflow limitation, lung function in symptom-free episodes, and severe hyperinflation [12]. If no clear distinction is possible and patients currently present with predominant features of COPD, i.e. relevant gas exchange impairment or hyperinflation, or signs/symptoms of chronic bronchitis or emphysema (if computer tomography scan available) [13], they are excluded from the study. Further exclusion criteria are: severe upper respiratory tract infection (URTI), or severe exacerbation during the last 4 weeks, ensuring that patients are in a stable phase of their disease [10, 11]. Controls had to be without any pulmonary disease but were allowed to have concurrent allergic rhinoconjunctivitis.

The overall study design of the DZL Asthma Cohort is shown in Fig. 3 a and b, and detailed later in the text. Healthy controls are seen at the study centres once, yet their age distribution will allow comparisons across the whole age range of recruited cases. For cases, the design includes a first study visit at time of recruitment and regular yearly follow-up study visits afterwards.

Before recruitment, in- and exclusion criteria for eligible individuals are assessed by participating centres. In the case of children, this is additionally performed with screening leaflets used by participating primary care physicians with parents being asked for consent to transfer contact data to study centres prior to any communication between study centres and interested families. After information about the study and informed consent by study participants (> 8 years of age) and, if necessary, all care-takers, the study phase starts with recruitment and the first study visit of cases in each study centre. On this occasion, lung function measurements are performed, and questionnaires are used to assess important clinical and epidemiological data. Furthermore, extensive biomaterial collections are implemented (see Fig. 3 a and b for an overview over the time flow of recorded data as well as tests and procedures).

Recruitment of new cases for the paediatric arm of ALLIANCE is on-going. From September 2013 until and including December 2016, a total of N = 540 children (n = 415 cases, thereof n = 209 preschool wheezers and n = 206 childhood asthmatics, n = 125 healthy controls) were recruited as eligible study participants. For the adult arm, N = 255 study participants (n = 208 cases, n = 47 healthy controls) were included until and including December 2016 (see Fig. 2).

After 12 months, all cases are invited for the first follow-up study visits at the study centres with identical measurements and data collection as during the baseline visit. On this occasion, a follow-up questionnaire is used to reassess important clinical and epidemiological items, likewise for all consecutive follow-up visits. In children, equivalent follow-up visits are performed every 12 months, while in adults, the schedule for deep-phenotyping follow-up visits is stretched.

Data collected during follow-up of cases has been added to the central database, quality controlled, and is reported here. In contrast to children, primary recruitment of adults was stopped in 2017 and only continued for those individuals who are already participants of the paediatric arm and turn adult during the time-course of the study.

Until the end of 2016, there was reasonable attrition during follow-up of cases (children and adults) as part of ALLIANCE (see Table 2). Among eligible children, i.e. individuals recruited until and including 2015 and with possible follow-up until and including 2016, we lost n = 52 of n = 324 children (16.0%) that should have completed study visits 12 months after initial recruitment, while this was the case for n = 17 of n = 172 (9.0%) 24 months, and for n = 4 of n = 20 (20.0%) 36 months after initial recruitment, respectively. Among eligible adults, n = 16 out of n = 169 (9.5%), who should have completed study visits 12 months and n = 3 out of n = 81 (3.7%), who should have completed study visits 24 months after initial recruitment, were lost over time. All individuals lost to follow-up were due to personal reasons. Further attrition during follow-up in 2017 and upcoming years will be evaluated regularly. Demographics of the N = 795 study participants of the DZL ALLIANCE cohort as part of the ALLIANCE database (at baseline for cases, only including eligible study participants) are displayed in Table 3 for both children and adults.

Data are derived from four sources: (a) hospital and medical records (used to extract information about wheeze and asthma diagnoses as well as in- and exclusion criteria), (b) questionnaires and structured interviews, (c) telephone interviews, and (d) objective measurements. A detailed description displaying the data collected in ALLIANCE for children and adults is presented in Table 1.

Questionnaires are used to collect information on health conditions, with an emphasis on respiratory and atopic symptoms, infections, and sociodemographic and environmental exposures. In children, a baseline questionnaire further collects information on pre- and perinatal risk factors including family past medical history. During follow-up, a further questionnaire assesses the study participants’ history during the period prior to the study visit. Both questionnaires used in the paediatric arm contain previously validated questions from the International Study of Asthma and Allergies in Childhood (ISAAC) [14]. Questionnaires in the adult arm cover the same topics and adopted the wording of the questions in an appropriate way. However, while some sections, e.g. pre- and postnatal risk factors, had to be shortened due to recall bias, other questions e.g. on comorbidities and co-medication were included and further validated questionnaires are distributed separately (Asthma Control Test, ACT [15]; Asthma Control Questionnaire, ACQ [16]; Asthma Quality of Life Questionnaire, AQLQ [17]; Multidimensional Fatigue Inventory, MFI-20 [18]). All questionnaires are used with little variation since 2012. For children, all questionnaires are programmed as an online database for data entry during interview. For adults, print-out versions of the structured questionnaires are primarily filled in by the patient and re-checked by the responsible study physician. Additional telephone interviews are used within a time window of up to one week after scheduled meetings to collect epidemiological data with use of baseline or follow-up questionnaires whenever these could not be completed during study visits.

For ALLIANCE participants, objective measurements include routine lung function tests such as spirometry and bodyplethysmography, but also more specialized techniques such as single and multiple breath washout measurements (SBW and MBW, respectively) [19] to assess small airway function and ventilation inhomogeneity. This is complemented by qualitative and quantitative assessments of airway inflammation using exhaled nitric oxide (eNO) measurements [20] as well as analyses of exhaled breath with an electronic nose (e-nose, in children only) [21] and with gas chromatography – mass spectrometry (GC/MS) [22]. In adults, assessment of small airway function is additionally performed by impulse oscillometry (IOS) [23] and capnovolumetry [24]. Objective lung function measurements in adults are further complemented by whole body bioimpedance measurement and objectively measured daily physical activity [25‐27]. All measurements are performed according to current guidelines by the European Respiratory Society (ERS) and the American Thoracic Society (ATS) [19, 20, 28‐33].

For ALLIANCE study participants and healthy controls, biomaterial collection during study visits include: peripheral blood samples (inflammatory markers, cell subpopulations and cytokines, sensitization pattern, metabolomics, proteomics (in children only), lipidomics, genomics, transcriptomics, and epigenomics); skin, nasal, pharyngeal, and nasopharyngeal swabs (microbiome, virome); collection of nasal secretions (cytokines); primary epithelial cell scrapings (transcriptome, primary epithelial cell culture); induced sputum samples (cytokines, microbiome, cell subsets); and urine (metabolomics); as well as stool samples (microbiome) only in children. Parents of participating children are also asked for a blood sample (genomics).