3.2 Performance of ELISA serological tests using r-PROE and r-IGLL1
The immunization against r-PROE and r-IGLL1 of BFL, FDL patients and control subjects was presented on the boxplots of Figure 1. As shown in Figure 1A, BFL patients (median: 1.24) had significantly higher levels of antibodies to r-PROE than FDL patients (median: 0,66, K-test p-value: 2.2 x 10-5) as well as controls exposed (median :0.34, K-test p-value: 1x 10-5) and unexposed to feathers (median: 0.37, K-test p-value: 1 x10-5).
In addition, FDL patients had significantly higher antibody levels than controls exposed (K-test p-value: 2.1 x10-5) and unexposed to feathers (K-test p-value: 9.8 x10-5). On the other hand, as shown in Figure 1B, the level of antibodies to r-IGLL1 was significantly higher in BFL patients (median: 2.36) compared to FDL patients (median: 0.33, K-test p-value: 4.2 x 10-7), controls exposed (median: 0.19, K-test p-value: 6.7 x 10-6) and unexposed to feathers (median: 0.24, K-test p-value: 6.7 x 10-6). However, no-significant difference using kruskalmc between FDL patients and controls unexposed to feathers were found (K-test p-value: 0.06). Consequently, r- IGLL1 was contributive to the diagnosis for the serological diagnosis of patients with BFL but ineffective to support FDL diagnosis.
The characteristics of the r-PROE and r-IGLL1 ELISA (sensitivity, specificity and area under the curve) performed by the ROC curve analysis were presented in Table 2. The ELISA test using r-PROE with an index threshold of 0.5 has shown that 74.2% of patients diagnosed with FDL have a positive test compared to 86.7% of controls with a negative test (AUC=0.9). These proteins were effective for the diagnosis of both chronic and acute forms. Indeed, the r-PROE ELISA test provided a consistent diagnosis for 15/20 patients suffering from chronic FDL and 8/11 patients suffering from acute FDL. The ELISA test based on the use of the r-IGLL1 protein provided a correct result for 6/6 patients with chronic BFL and 8/9 patients with acute BFL.
In the case of exposure to both feather bedding and birds, analyses of the ROC curve showed a significant differential threshold between FDL and BFL cases. If the r-PROE ELISA test index value was between 0.5 and 1, the interpretation was in favor of FDL, while the “bird” etiology was preferred for an index value greater than 1 (Table 2). In addition, if the bird is the cause of the disease, the index value of the IGLL1-ELISA test will be greater than 1.1. Based on these results, we have proposed a key choice to support the diagnosis of HP of avian origin according to the patient’s exposure, in Figure 2.
DISCUSSION AND CONCLUSIONS
The development of tools to improve the diagnosis of HP is a challenge and at the heart of current concerns, as HP experts have highlighted the need for a diagnostic test to measure serum IgG levels with a well-accepted threshold2. In the present study, we showed that an ELISA test using r-PROE allowed effective discrimination between 31 FDL patients and 30 controls. The r-IGLL1 ELISA test was only useful for the serological diagnosis of the 15 BFL patients. These results will serve as a guide for the clinicians in the choice and interpretation of serological tests to be performed according to the type of avian exposure of patients.
HPs are often difficult to diagnose in part because of the difficulty to identify the antigenic source but also because the clinical behavior of these diseases mimics those of other pulmonary diseases. Indeed, despite thorough investigations, the offending HP-antigen was not identified in 25-53% of cases17, 18. The group of Barcelona reported that 43% of patients initially identified with idiopathic pulmonary fibrosis actually had a chronic form of HP, half of these cases were due to exposure to feather bedding9. Thus, exposure to avian feather proteins may be an unrecognized cause of HP7, 19, 20. The detection of antigen-specific IgG antibodies is useful to support the diagnosis of HP, as it allows identification of the causal antigen2, 12. In our experience, serological analyses were mainly used to rule out the diagnosis of HP in favor of other respiratory pathologies21, but also to identify the etiologic agent involved in HP. For serological analyses, several methods to determine precipitins (Ouchterlony double diffusion and Immunoelectrophoresis)22 or specific IgG antibodies (ELISA and ImmunoCAP®, Uppsala, Sweden) have been used in analytical laboratories23.
ELISA was described as being more sensitive than precipitin assays in detecting antibodies to pigeon droppings for BFL serodiagnosis13, 22. Currently, the antigens used for the serological diagnosis of FDL are purified (commercial or non-commercial) from goose feathers, duck feathers, a mixture of both, or from those of other bird species (pigeon, parakeet)24, 25. Several studies have shown significantly high antibody levels in patients using feather duvet and pillow antigens than controls24-26. Comparison of the data obtained is difficult due to the different techniques used but also to the lack of standardization of antigen production11.
New techniques, such as proteomics coupled with mass spectrometry (LC-MS/MS), now make it possible to characterize proteins associated with the disease. IgG antibodies against r-IGLL1 and r-PROE proteins have been identified as biomarkers of BFL and have been found in droppings, bloom, and pigeon serum using an optimal immunoproteomic approach13, 14.
The diagnostic performance of an ELISA test for BFL performed with r-IGLL1 and r-PROE gave the best specificity (100%) and sensitivity (84%)13. These proteins are involved in the immune and digestive systems of birds13. Several studies have suggested the presence of cross-reactive antigenic reactions between different bird species or different avian matrices, especially for pigeons 14, 27-29. Recently, significant correlations have been found in serological analyses between pigeon, duck, and goose antigens11. Based on the BLASTp alignment, our results showed that the amino-acid sequence of duck IGLL has higher identity with goose IGLL (ID: 81%) than pigeon IGLL1 (ID: 65%) (Supplemental data). On the contrary, the amino-acid sequence of duck PROE has higher sequence identity with pigeon PROE (81%) than with the orthologous protein in geese (73%). The conservation of the amino-acid sequences of these proteins is a contributing factor to the antigenic cross-reaction observed in serological analyses14, 28. In the present study, we evaluated the performance of an ELISA using the pigeon r-IGLL1 and r-PROE proteins to support the serological diagnosis of FDL cases.
We found the ELISA test using r-IGLL1 to be useful to support the diagnosis of BFL patients but not for FDL cases. Conversely, we found significantly higher levels of circulating IgG antibodies against r-PROE in BFL and FDL patients than controls. Indeed, r-PROE was the most effective antigen for discriminating FDL patients from controls exposed and unexposed to feathers. The characteristic performance of the r-PROE ELISA test using an optimal threshold index value was as follow: sensitivity of 74.2%, specificity of 86.7% and AUC of 0.9.
The antibodies of patients directed against r-IGLL1 and r-PROE are respectively two and seven times significantly higher in BFL patients compared to FDL patients. Although patients were exposed for long periods to their pillows, but with little agitation, the amount of inhalable antigenic protein was probably lower than that inside an aviary (66.6% of our BFL patients were pigeon breeders). Indeed, in such a location, antigens come from droppings, bloom, and feathers and are frequently suspended by the birds or the breeder during cleaning. Such differences in exposure intensity may explain the higher level of antibodies in BFL patients compared to the FDL observed in our study.
Since the intensity of sensitization is significantly different between BFL and FDL patients against r-PROE in ELISA (r-IGLL1 specific for BFL patients), we proposed a useful key for cases of patients exposed to both feathers and birds. Although r-PROE and r-IGLL1 were both effective for serodiagnosis of BFL cases, to simplify procedures, we recommend that only r-IGLL1 be used for the diagnosis of patients exposed to birds. Likewise, in the event of a suspected FDL case, r-PROE was the only protein to be used for the ELISA test. In case of multi-exposure (feather bedding + birds), an index value for r-PROE between 0.5 and 1 is in favor of the diagnosis of FDL, and above 1, in favor of BFL case.
Several studies have shown that FDL cases are related to childhood exposure to birds, and that contact with pillow feathers is a trigger for sensitization that occurred long before29-31. The use of a feather pillow/duvet should be discouraged in cases of BFL because continuous contact with avian antigens can induce disease progression and cause permanent lung damage9, 32, 33.
Finally, as there is no effective treatment to reverse the lung damage caused by the disease, early identification of the antigenic source is necessary. The diagnosis of FDL should be based on a proactive approach to find the antigen source to remove it from the patient’s environment21.
In conclusion, we recommend the use of r-PROE and r-IGLL1 proteins, respectively, for the serological diagnosis of patients exposed strictly to bedding feathers or birds and presenting with respiratory symptoms. These two ELISA tests allow the diagnosis of both chronic and acute forms of FDL and BFL cases. The ELISA test based on the use of r-PROE showed a sensitivity of 74.2% and a specificity of 86.7% for an AUC of 0.9 for FDL patients. The use of recombinant proteins guarantees highly standardized production and optimal inter-batch reproducibility.
The use of the ELISA test reduces the time taken to report results to patients / clinicians to 3 days. This time saving allows the implementation of an early avoidance strategy favorable to the patient’s state of health. This serological approach is efficient, standardized, fast, inexpensive and easy to implement.
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