ABSTRACT
Background : Feather duvet lung (FDL) is an underestimated form of acute and chronic hypersensitivity pneumonitis. Serological tests for FDL need to be validated. We investigated the ability of recombinant pigeon Proproteinase E (r-PROE) and Immunoglobulin-lambda-like-polypeptide-1 (r-IGLL1) proteins to support the serological diagnosis of FDL, and propose them as a serological tool for clinicians to differentiate cases from FDL and Bird fancier’s lung (BFL).
Methods: Specific IgG antibodies against r-PROE and r-IGLL1, analyzed with ELISA, were measured in patients diagnosed with FDL (n=31), BFL (n=15) controls exposed (n=15) and unexposed to feathers (n=15).
Results: The sensitivity and specificity of the r-PROE ELISA for the serological diagnosis of FDL cases versus exposed and unexposed controls were 74.2% and 86.7% respectively, with an index threshold of 0.5. (AUC: 0.9). In addition, this serological test was effective to support the serological diagnosis of FDL and BFL cases with significantly different thresholds. The r-IGLL1 ELISA was only effective for the serological diagnosis of BFL. Also, these two serological tests were useful for the diagnosis of both chronic and acute forms.
Conclusions: The new diagnostic test for FDL using r-PROE protein should help to detect overt and hidden cases of FDL. The combination of both test will help the clinician in distinguish between the etiology of birds or feathers duvet.
KEY WORDS : Diagnosis, ELISA, Hypersensitivity pneumonitis, Pillow, Specific IgG antibodies
Introduction
Hypersensitivity pneumonitis (HP) is an inflammatory and/or fibrotic diffuse parenchymal lung disease, arising in susceptible individuals after repeated and prolonged inhalation to specific antigens1. The inciting antigens are most often derived from bacteria, fungi, animal, and plant (glyco) proteins, low molecular-weight chemicals, and metals2. The diagnosis of HP is challenging given its heterogeneous clinical presentation and overlapping features with many other forms of interstitial lung disease. In general, its diagnosis is based on a combination of clinical, radiological, histological, and biological features1, 3, 4.
One of the most common forms of HP is bird-fancier’s lung (BFL), caused by exposure to wild or domestic birds5. Antigen sources are bird droppings, feathers and bloom (a waxy powder that coats the feathers)6. However, exposure to feather/down proteins hidden in commonly used objects is involved in another group of avian HP called feather duvet lung (FDL). Feather pillows and down duvets are the main antigenic sources due to their proximity to the respiratory tract and long duration of daily exposure. An increase in the use of goose and duck down in bedding, decorative pillows, clothing and stuffed furniture has been observed in recent years7. Exposure to feathers from bedding has been assessed at 30% of the population in Catalonia 8 and France (personal data). In Catalonia, the prevalence of FDL for a period of 10 years has been estimated at 6.2 / 100,000 users of feather bedding compared to a prevalence of 54.6 / 100,000 among bird breeders8 . FDL is characterized as an under-recognized and consequently underestimated form of HP in several studies7, 9, 10. In some cases, if not diagnosed early enough, the disease can progress to irreversible pulmonary fibrosis, leading to permanent damage and the premature death of the patient2.
A recent modified Delphi survey on chronic HP shows that exposure to a causative antigen is the most important clinical variable supporting a confident diagnosis3. Although the use of serological tests is not universally accepted, they can be used to demonstrate antigenic exposure by looking for circulating precipitins or IgG antibodies3, 11, 12.
The antigens routinely used in the diagnosis of FDL are purified mixtures of goose or duck feather whose performance varies from one batch to another. The identity of the antigens involved in FDL is currently unknown. Recent studies have characterized two pigeon proteins called immunoglobulin-lambda-like-polypeptide-1 (IGLL1) and proproteinase E (PROE)13, which are useful for the serological diagnosis of BFL13, 14.
The IGLL1 protein has been identified in droppings, bloom, and pigeon serum and PROE protein in droppings and bloom. A strong correlation has been shown between the IgG antibody response of patients sensitized to pigeon, duck and goose antigens11. Therefore, we hypothesize that amino-acid sequences close to those found in IGLL1 and PROE are part of proteins from the feathers (goose, duck) used for the manufacture of pillows and duvets.
This study aimed (i) to assess the performance characteristic of r-IGLL1 and r-PROE (ii) to support the serological diagnosis of FDL, compare the level of sensitization of FDL and BFL patients and (iii) to provide an effective and useful tool for clinicians to guide the diagnosis of HP of avian origin.
  1. METHODS
  2. Study design
This study comprised patients diagnosed with FDL, BFL and controls exposed or unexposed to feathers without HP. The 31 FDL patients were recruited in the Pneumology Unit at the Hospital Universitari Vall d’Hebron (Barcelona, Catalonia, Spain). The 15 BFL patients and the 30 controls were recruited by the University hospital of Besancon (France). The patients who are included in the study have given their informed consent. The FDL and BFL patients had a confident diagnosis of HP according to the criteria reported below. Blood collection was performed for each patients and controls at the time of diagnosis and kept frozen to -80°C until their analysis.
Hospital Universitari Vall d’Hébron (Barcelona, Catalonia, Spain)
The criteria used to diagnose HP were those defined by Vasakova et al.12. FDL patients (n=31) were selected from an ongoing prospective study undertaken to evaluate the exposure of environmental factors as potential causative factor/s for new onset interstitial lung disease (ILD). Patients were followed up long-term over the study period from January 2004 to December 2013. The diagnosis of FDL was done according to the following main criteria: (i) no exposure to birds or other environmental factors that could induce HP, (ii) Past or present exposure to feathers hidden in the environment. These last two criteria were assessed using a standardized questionnaire15, (iii) Positive specific inhalation challenge and/or specific IgG in the serum. The following antigenic panels were used to measure serum IgG levels by ELISA: bird feathers, bird serum (goose, pigeon, parrot, parakeet, canary), goose feathers and fungus (Aspergillus, Penicillium, Mucor). These patients were included in a previous study (Ethical Committee number: PR(AG) 165/2016), for more clinical details see reference 8.
Besançon University Hospital (France)
All BFL patients (n=15) were given a diagnosis between September 2010 to January 2016 according to the following criteria16 : (i) A well-known bird exposure (detailed questionnaire, positive precipitin serological test using crude antigens from bird droppings in relation to patient exposure, (ii) No past or present exposure to feathers or other environments involved in HP, (iii) Symptoms and High-resolution computed tomography (HRCT) compatibles with HP and basal crepitant rales, (iv) Bronchoalveolar lavage (BAL) lymphocytosis and (v) Decreased DLCO during exercise. This protocol of recruitment was approved by the local ethics committee (CPP-Est II 15/496). These 15 patients were selected according to their availability in serum from a cohort previously described13.
The control groups included 30 patients without HP. They were selected according to their exposure and classified into the following two groups:
Indirect ELISAs using r-PROE and r-IGLL1 were performed in April 2019, in the Parasitology-Mycology department as described by Rouzet et al.13. All analyses were performed on the same day for all patients, i.e. retrospectively to the diagnosis. ELISAs were performed without knowing the clinical status of the sera from the Vall d’Hebron department.
Briefly, the wells of 96-well plates (PolysorpImmunomodule, Nalge Nunc®, Rochester, UK) were coated by incubation with 100 µL of 10 µg/mL r-PROE and 5 µg/mL r-IGLL1 in phosphate-buffered saline (Sigma-Aldrich®, St Louis, USA) at 4°C overnight. Serum samples were diluted 1/100 in dilution buffer, 100 µL deposited in triplicate into the wells, and the plates incubated for 1 h at 37°C. Polyclonal rabbit antibodies (anti-IGLL1-PROE) (RD-Biotech®, Besançon, France) were used as a positive control of the test (1.8 µg/mL) and as a reference sample (0.4 µg/mL). The three optical density values (OD) were blank-corrected and averaged and the standard deviation and variation coefficient calculated. If the coefficient of variation of the triplicate was greater than 20%, the outlier was removed. The ELISAs were carried out twice. An index was calculated as follows: Index = mean OD of the blank-corrected sample replicates/mean OD of the blank-corrected reference sample.
Statistical analysis
All statistical analyses were performed with R 3.5.3 software and a p-value of 0.05 was used to define statistical significance. Receiver-operator characteristics (ROC) analysis (pROC-package) was performed to evaluate the ability of r-PROE and r-IGLL1 in ELISA to discriminate between Patients with FDL (status=1) and controls (exposed and unexposed to feathers, status=0). Therefore, BFL patients have been removed from ROC analysis to avoid overestimation of the performance of these 2 recombinant proteins. The performance of r-PROE and r-IGLL1 was also evaluated to detect the threshold discriminating cases of BFL (status=1) and FDL (status=0). The normality assumption for continuous variables was tested using the multivariate Shapiro-Wilks test; the data were not normally distributed. The nonparametric Kruskal-Wallis (K) test was used to compare the median values between the 4 following groups: FDL, BFL, Expo feathers and Unexposed to feathers. Following a significant K test, a multiple comparison post hoc test (KMC) using the kruskalmc function (“pgirmess”package) was performed to make inter-group comparisons.