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.
- METHODS
- 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:
- 15 subjects exposed to feather bedding (« Expo feathers »), but not
exposed to birds or other environment at risk of HP at the time of
diagnosis. They have already been sensitized (i.e., immune system
already stimulated by antigen) to avian antigens from feathers but
have not developed HP.
- 15 subjects not exposed to feather bedding (« Unexposed »), birds or
any environment involved in HP at the time of diagnosis or in the
past.
- Measuring of serum specific circulating IgG antibodies
against r-PROE and r-IGLL1
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.