Protein Estimation in Whole Lung Lavage Fluid in Hereditary Pulmonary Alveolar Proteinosis Due to a Novel GM-CSF Receptor Mutation.

Pulmonary alveolar proteinosis (PAP) is associated with abnormal surfactant accumulation in alveoli, leading to respiratory failure if untreated. It is classified as primary (sub-classified as autoimmune or hereditary), secondary, or congenital. Children with hereditary PAP (hPAP) have an inherited defect in the GM-CSF signaling pathway, required for surfactant clearance by macrophages. We describe a child with hPAP having a novel mutation, and suggest refinement of some management aspects. An 8.5-year girl presented with dry cough, progressive breathlessness, and poor weight gain for 2 years. She was confined to bed for 1 month before presentation. There was no history of fever, stridor, wheezing, syncope, palpitations, extrapulmonary infections, atopy, or allergen exposure. She was born at term, to nonconsanguineous, healthy parents. There were no neonatal problems and she had been well, before the current illness. Her younger siblings were also healthy. Clinical examination revealed tachypnoea, hypoxia in room air, severely malnourished state, absence of clubbing, and bronchial breathing over the left mammary area. There were no crackles or wheeze. Chest X-ray showed bilateral consolidation and computerized tomography (CT) scan revealed diffuse crazy-paving (Figure S1A,B). Anticipating the need for subsequent chest imaging, we performed simultaneous thoracic magnetic resonance imaging (MRI), to avoid future radiation exposure (Figure 1A). Short duration lung MRI was performed on 3T MRI scanner using T2 weighted turbo spin echo, balanced turbo field echo, and MultiVane XD sequences in the axial plane. The total scan time was 3.5 min. Bronchoalveolar lavage (BAL) fluid was milky in appearance (Figure S1C). Cytological examination showed foamy macrophages and abundant eosinophilic and PAS-positive granular material (Figure S1D). It did not yield bacteria, fungi, Mycobacteria, Pneumocystis jirovecii, or SARS-CoV-2. HIV serology, serum ANA and ANCA were negative. Lung biopsy confirmed the diagnosis of PAP (Figure S1E). Clinical exome sequencing showed a novel homozygous missense variation in exon 7 of CSF2RA gene [c.571T>G (p.Tyr191Asp)], hence categorized as “uncertain significance.” The child's parents declined genetic testing in themselves and their other children, therefore further characterization of the identified variation was not feasible. We also did not have facilities to estimate GM-CSF receptor expression on cell surface, or measure the effects of downstream signaling. However, serum GM-CSF was elevated to 203.0 pg/ml (lab normal ≤7.0 pg/ml), with normal anti GM-CSF autoantibody level (lab normal ≤3.1 mcg/ml), which is consistent with a GM-CSF receptor defect. hPAP secondary to mutation in alpha subunit of GM-CSF receptor was first described in 2008, and our patient had clinical, radiological and pathological features, similar to the handful of cases reported since then.1, 2 However, there are no previous reports with the mutation identified in the index case. In hPAP, therapeutic whole lung lavage (WLL) is efficacious, although the procedure is not fully standardized in children. Typically, one lung is selectively ventilated, and the other lavaged with aliquots of warm saline, until the effluent appears clear. After stabilizing the child, we performed WLL on both lungs (in separate sessions) under general anaesthesia. In the first session, the right lung was lavaged using 100 ml aliquots until the effluent was grossly clear (Figure 2A). After the procedure was completed, we measured the protein level in each aliquot of effluent, by spectrophotometry. We observed sequential reduction of protein level from 310.6 mg/dl in the first aliquot, to 31.7 mg/dl and 37.3 mg/dl in the last two clear-appearing aliquots (Figure 2B). Although the patient had reduction in respiratory distress, oxygen dependence persisted. An MRI done 48 h after WLL showed marked reduction in the extent and intensity of consolidation on the right side (Figure 1B). Seven days later, WLL was performed on the left lung, using 200 ml aliquots. This time, in addition to visual inspection of the effluent for clearance, protein concentration was measured in each aliquot, targeting a final value between 5 and 15 mg/dl, which has been reported as the normal range of BAL protein in children with healthy lungs.3, 4 Therefore, although the effluent cleared by the seventh aliquot and protein level showed marked reduction from 400.5 mg/dl in the first aliquot to 22.2 mg/dl (Figure 2C), WLL was continued till the last three aliquots showed protein levels of 10.3, 9.6, and 6.7 mg/dl (Figure 2D). Following left lung lavage, the child improved dramatically, and oxygen could be omitted completely. MRI after 48 h showed radiological resolution on the left side (Figure 2C). The child was discharged, and remained well. Follow-up MRI after 2 months showed no radiological progression (Figure 2D). Our experience suggests that real-time measurement of BAL effluent protein can be a simple, point-of-care, objective method to guide WLL, especially its cessation. Ideally, surfactant levels should be measured, including the phospholipid content as well as specific surfactant proteins. Our institution lacks these facilities. We understand that most institutions across the world also lack facilities for these analyses in real-time during the WLL procedure. We suggest that BAL effluent protein estimation is a feasible and acceptable surrogate. However, there is very limited data about the BAL protein content in normal lungs of children. Two small studies reported values of 10.8 ± 3.9 mg/dl and 5–15 mg/dl,3, 4 hence we targeted this range. There is also no data on whether these levels can be achieved with WLL in children. However, one study reporting kinetics of protein washout during WLL in children with PAP of unknown cause, showed similar results to ours.5 Most of the currently available literature on radiological imaging in PAP is centered around CT scanning, both for the initial diagnosis and subsequent monitoring. We have been attempting to use MRI in children with diverse respiratory conditions, to minimize radiation exposure. We were gratified to observe that in the index case, MRI images correlated with the clinical improvement. More studies are required to assess whether MRI may be an efficacious imaging modality in hPAP. In conclusion, we propose two novel approaches for the management of childhood PAP, viz. real-time measurement of BAL effluent protein to objectively guide whole lung lavage, and MRI as a potential alternative to CT scan for diagnosis, treatment response, and follow-up. Joseph L. Mathew: conceptualization (equal); investigation (lead); methodology (lead); supervision (lead); validation (equal); writing—review and editing (lead). Ketan Kumar: conceptualization (equal); writing—original draft (lead). Kamal K Singhal: conceptualization (supporting). Preethy J Mathew: methodology (equal); resources (equal). Anmol Bhatia: investigation (equal); methodology (equal). Amanjit Bal: investigation (equal); resources (equal); writing—review and editing (supporting). Nalini Gupta: investigation (equal); methodology (equal); writing—review and editing (supporting). Prema Menon: investigation (supporting). meenu singh: supervision (equal); validation (equal). We acknowledge Dr. Bruce C. Trapnell at Cincinnati Children's Hospital, and his team for sharing their expertise and advising with the diagnostic workup. We thank Dr. Sachin Singh, Dr. Anvesh Reddy, Dr. Radhika Srinivasan and Dr. Sandhya Yaddanapudi at PGIMER, Chandigarh for their valuable contribution in managing the child. The authors declare no conflicts of interest. A written informed consent was obtained from the child's father for publication of the case report and images included in the manuscript. The Departmental Review Board approved the submission. Data sharing not applicable—no new data generated. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.