Lipid Accumulation and Drug-Induced Interstitial Lung Disease Associated with Amikacin Liposomal Inhalation Suspension in Patients with Mycobacterium avium Complex Lung Disease: A Singlecenter Retrospective Cohort Study

Lipid Accumulation and Drug-Induced Interstitial Lung Disease Associated with Amikacin Liposomal Inhalation Suspension in Patients with Mycobacterium avium Complex Lung Disease: A Singlecenter Retrospective Cohort Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lipid Accumulation and Drug-Induced Interstitial Lung Disease Associated with Amikacin Liposomal Inhalation Suspension in Patients with Mycobacterium avium Complex Lung Disease: A Singlecenter Retrospective Cohort Study Hiroki Wakabayashi, Kazutoshi Isobe, Nobuyuki Hiruta, Shusuke Kasuya, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8234124/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Mar, 2026 Read the published version in BMC Pulmonary Medicine → Version 1 posted 12 You are reading this latest preprint version Abstract Background Amikacin liposomal inhalation suspension (ALIS), an inhaled aminoglycoside, can effectively treat refractory or relapsed Mycobacterium avium complex lung disease (MAC-LD). However, ALIS is associated with drug-induced interstitial lung disease (DI-ILD), and underlying pathological mechanisms for the same are unclear. This study aimed to determine the aetiology and pathological characteristics of ALIS-associated DI-ILD and assess the impact of ALIS on pulmonary pathology. Methods This retrospective cohort study included 25 patients with MAC-LD who received ALIS treatment between September 2021 and October 2024. Clinical data, laboratory findings, high-resolution chest computed tomography results and pathological findings from transbronchial lung biopsy, surgical lung resection and bronchoalveolar lavage were evaluated. Results Of the 25 patients, 8 developed DI-ILD after a mean duration of 98.5 ± 159.8 days following ALIS initiation. Histopathological evaluation showed infiltration of enlarged foamy macrophages containing lipid deposits in patients with and without DI-ILD. In patients with DI-ILD, bronchoalveolar lavage fluid exhibited inflammatory cell infiltration; however, these findings were not diagnostic for a specific entity. Serum Krebs von den Lungen-6 levels significantly increased from baseline to DI-ILD diagnosis (407 ± 231 vs. 434 ± 250 U/mL; P = 0.0422), whereas eosinophil count, C-reactive protein and lactate dehydrogenase levels did not change significantly. Seven of eight patients with DI-ILD exhibited clinical improvement following ALIS discontinuation or dose reduction; one patient required corticosteroid therapy. Of the 8 patients, 5 continued inhalation therapy by switching to every-other-day dosing. The sputum culture conversion rate was maintained even in patients with DI-ILD (63% vs. 47%; P = 0.673). Conclusion Lipid deposition within the lungs was observed irrespective of DI-ILD, despite no pathognomonic findings being identified for ALIS-associated DI-ILD. Some patients continued therapy by reducing the dosing frequency while preserving therapeutic efficacy even when DI-ILD occurred. Amikacin bronchoalveolar lavage drug-induced interstitial lung disease exogenous lipoid lipoid pneumonia Mycobacterium avium complex pneumonia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Background Mycobacterium avium complex lung disease (MAC-LD) is a chronic pulmonary infection caused by nontuberculous mycobacteria, predominantly Mycobacterium avium complex (MAC) [ 1 ]. In Japan, the clinical burden of MAC-LD has been increasing since 2010, surpassing the incidence of new tuberculosis cases in 2018 [ 2 ]. Despite long-term multidrug regimens, MAC-LD management remains challenging owing to frequent relapse and treatment refractoriness [ 1 ]. According to international guidelines, the standard treatment regimen includes macrolides, ethambutol and rifamycin, with aminoglycosides added for patients with severe or refractory disease [ 1 ]. Injectable aminoglycosides, such as amikacin, streptomycin and kanamycin, have been used traditionally; however, in 2020, amikacin liposomal inhalation suspension (ALIS) was approved by the US Food and Drug Administration as an alternative [ 1 , 3 ]. ALIS is indicated for patients with refractory or relapsed MAC-LD; it has demonstrated efficacy when combined with guideline-based therapy, achieving sputum culture conversion in approximately 29% of refractory cases [ 4 ]. Compared with intravenous aminoglycosides, ALIS produces lower serum concentrations, potentially minimising the risk of ototoxicity, nephrotoxicity and vestibular toxicity while preserving antimicrobial efficacy [ 5 – 7 ]. Despite these advantages, ALIS has been associated with specific adverse events such as hoarseness and drug-induced interstitial lung disease (DI-ILD) [ 3 ]. The CONVERT study, an international phase 3 open-label trial, reported hypersensitivity pneumonitis in 3.1% of patients receiving ALIS, which led to treatment discontinuation [ 3 ]. In addition, several case reports have described the occurrence of interstitial pneumonia in ALIS-treated patients [ 8 , 9 ]. However, the pathophysiological mechanisms underlying ALIS-associated DI-ILD remain poorly understood. This study aimed to investigate the aetiology of ALIS-associated DI-ILD by evaluating the clinical characteristics, laboratory results, high-resolution chest computed tomography (HRCT) findings and pathological findings of patients with MAC-LD receiving ALIS treatment. 2. Methods 2.1. Study Design This single-centre retrospective cohort study was conducted at Toho University Medical Center, Sakura Hospital. The study protocol was approved by the hospital’s institutional ethics committee (approval number: S23058_S23006). The requirement for informed consent was waived owing to the retrospective nature of the study. Instead, the study details were publicly available on the institutional website, allowing potential participants to opt out. 2.2. Patient Population The study included patients diagnosed with MAC-LD based on the 2020 ATS/ERS/ESCMID/IDSA clinical practice guidelines who received ALIS treatment between September 2021 and October 2024. Patients with an observation period of at least 6 months after the ALIS initiation were considered eligible. 2.3. Data Collection Patient data, including age, sex, body mass index (BMI), negative sputum conversion status, treatment duration, and DI-ILD development, were extracted from electronic medical records. Negative sputum conversion was defined as three consecutive negative sputum cultures that are collected at intervals of at least one month. The date of sputum culture conversion was assigned as the date on which the first of these three negative samples was submitted. The conversion date, for patients in whom sputum production resolved with treatment and samples could no longer be submitted, was defined as the first outpatient visit at which sputum could not be provided. Furthermore, spirometry as well as laboratory, HRCT and pathological findings from transbronchial lung biopsy (TBLB), bronchoalveolar lavage (BAL) and surgical lung resection for treatment (SLR) were reviewed. 2.4. ALIS Administration All patients received 590 mg of ALIS (Arikayce; Insmed Inc., Bridgewater, NJ) administered using the eFlow® rapid nebuliser system (Lamira; PARI Pharma GmbH, Munich, Germany). All patients were trained on proper inhalation techniques during a 3–7-day hospitalisation period or at least three outpatient visits. Inhalation adherence and technique were evaluated monthly by respiratory specialists and trained nurses. 2.5. Diagnostic Criteria for ALIS-Associated DI-ILD Due to the absence of established diagnostic criteria for ALIS-associated DI-ILD, the diagnosis in this study was based on the following conditions: (1) appearance of new bilateral consolidations or ground-glass opacities on chest computed tomography (CT) following ALIS initiation, which improved with dose reduction or treatment discontinuation; (2) presence of at least one symptom, such as fever, cough or dyspnoea; (3) negative PCR test for severe acute respiratory syndrome coronavirus 2; (4) no newly introduced medications within 1 month before symptom onset; (5) no history of autoimmune disease or HIV/AIDS; (6) no use of corticosteroids or other immunosuppressive agents; and (7) other causes, such as bacterial pneumonia, viral infections (e.g., influenza), heart failure, and progression of MAC-LD, were excluded based on clinical course, microbiological tests, and imaging findings. The date of DI-ILD diagnosis was defined as the time when abnormal findings were first identified on chest CT. Two thoracic radiologists and two pulmonologists independently reviewed CT scans to reach a final diagnosis of DI-ILD. 2.6. Laboratory Evaluation At ALIS initiation and DI-ILD onset, the levels of serum biomarkers associated with interstitial lung disease—C-reactive protein (CRP), lactate dehydrogenase (LDH), Krebs von den Lungen-6 (KL-6) and eosinophil count—were measured. Furthermore, the estimated glomerular filtration rate (eGFR) was evaluated. 2.7. Chest CT Assessment HRCT was performed before and after ALIS initiation to assess pulmonary involvement. Baseline disease severity was scored using a previously validated CT scoring system [ 10 ]. 2.8. Pathological Assessment Lung tissue from TBLB was fixed in 10% neutral buffered formalin, and SLR specimens were fixed using bronchial perfusion and immersion methods. Tissue processing involved sequential infiltration with 10%, 15% and 30% sucrose solutions at 4°C, followed by embedding in Tissue-Tek® optimal cutting temperature compound and then freezing in liquid nitrogen. Tissue sections were cut at 5–6-µm thickness for hematoxylin and eosin and Oil Red O staining when foamy macrophages were observed. BAL fluid samples were centrifuged, with the smears similarly fixed and stained. 2.9. Statistical Analysis Categorical variables were expressed as numbers and percentages, whereas continuous variables were expressed as means ± standard deviation (SD). Categorical variables were compared using the chi-squared or Fisher’s exact test, as appropriate, whereas continuous variables were compared using the Mann–Whitney U test. Paired t -tests were used to assess changes in biomarkers between baseline and the time of DI-ILD diagnosis. Analyses were conducted using the SPSS software (version 25; IBM Corp., Armonk, NY, USA), and P ≤ 0.05 was considered to indicate statistical significance. 3. Results 3.1. Study Population The study flow is shown in Fig. 1 . A total of 32 patients with MAC-LD who received ALIS treatment between September 2021 and October 2024 were initially enrolled in the study. Of them, seven were excluded as they had an observation period of < 6 months after ALIS initiation. Consequently, 25 patients were included in the final analysis, of whom 8 (32%) developed DI-ILD (DI-ILD group) and 17 (68%) did not (non-DI-ILD group). During ALIS treatment, three patients in the DI-ILD group underwent TBLB and BAL, whereas three in the non-DI-ILD group underwent SLR. The patients’ baseline characteristics are summarised in Table 1 . The mean age of the cohort was 74 ± 7.2 years. Of the included patients, 16% were male and 84% were female. The mean BMI was 18.1 ± 2.7 kg/m². No statistically significant differences were observed between the groups in pulmonary function parameters, sputum culture conversion rate or chest CT scoring. However, the DI-ILD group had a significantly lower eGFR than the non-DI-ILD group (62.5 ± 12.8 vs. 78.9 ± 13.3 mL/min/1.73 m²; P = 0.010). Table 1 Baseline Characteristics of the Study Population Characteristic All Patients (n = 25) DI-ILD Group (n = 8) Non-DI-ILD Group (n = 17) P -value Age (years), mean ± SD 74 ± 7.2 73 ± 5.2 74 ± 8.2 0.608 Sex, n (%), F/M 21/4 (84/16) 8 /0(100/0) 13/4 (76/24) 0.269 BMI (kg/m²), mean ± SD 18.1 ± 2.7 18.2 ± 1.6 18.1 ± 3.1 0.932 Negative sputum conversion, n (%) 13 (52) 5 (63) 8 (47) 0.673 Treatment duration (days), mean ± SD 404 ± 257 285 ± 158 460 ± 278 0.114 %FEV 1 , mean ± SD 92.6 ± 25.0 76.7 ± 9.0* 100.5 ± 26.8† 0.052 %FVC, mean ± SD 86.5 ± 23.0 77.2 ± 15.4* 91.2 ± 25.3† 0.237 CT score, mean ± SD 14.2 ± 4.7 14.4 ± 3.6 14.1 ± 5.3 0.880 CRP (mg/dL), mean ± SD 1.38 ± 2.29 1.11 ± 2.52 1.51 ± 2.24 0.708 LDH (U/L), mean ± SD 195 ± 43 185 ± 19 201 ± 51 0.413 KL-6 (U/mL), mean ± SD 494 ± 240 472 ± 247 507 ± 245 0.746 eGFR (mL/min/1.73 m²), mean ± SD 73.2 ± 15.1 62.5 ± 12.8 78.9 ± 13.3 0.010 Eosinophil count (/µL), mean ± SD 171 ± 168 189 ± 178 163 ± 169 0.839 a * (n = 6), † (n = 12) b Abbreviations : SD = standard deviation; BMI = body mass index; CRP = C-reactive protein; DI-ILD = drug-induced interstitial lung disease; eGFR = estimated glomerular filtration rate; FEV 1 = forced expiratory volume in 1 second; FVC = forced vital capacity; KL-6 = Krebs von den Lungen-6; LDH = lactate dehydrogenase 3.2. Clinical Course of DI-ILD The clinical courses of the eight patients diagnosed with ALIS-associated DI-ILD are illustrated in Fig. 2 . The mean duration from ALIS initiation to DI-ILD diagnosis was 98.5 ± 159.8 days. Following DI-ILD onset, seven of the eight patients exhibited radiologic and symptomatic improvement after either treatment discontinuation or adjustment to alternate-day dosing. The mean duration from DI-ILD diagnosis to radiologic improvement on chest CT was 43 ± 32 days. Remaining one patient required oral corticosteroid therapy owing to persistent symptoms. Notably, of the 8 patients, 5 (63%) continued ALIS by reducing the dosing frequency to every-other-day inhalation, whereas 3 achieved negative sputum conversion after reducing the dosing frequency. 3.3. Laboratory Findings Changes in serum CRP, KL-6 and LDH levels as well as eosinophil count from baseline to DI-ILD diagnosis are presented in Fig. 3 . No significant changes were observed in CRP, LDH or eosinophil count between the two time points. However, KL-6 levels significantly increased at DI-ILD diagnosis compared with that at baseline (407 ± 231 vs. 434 ± 250 U/mL; P = 0.0422). 3.4. Representative DI-ILD Case Findings Representative chest CT and pathological findings from Patient #3—the only case requiring corticosteroid therapy—are presented in Fig. 4 . A baseline CT scan performed before the ALIS initiation revealed no evidence of DI-ILD (Fig. 4 A). The patient experienced cough and dyspnoea on day 444 after ALIS initiation. HRCT showed ground-glass opacities with interlobular septal thickening, raising suspicion for ALIS-associated DI-ILD (Fig. 4 B); hence, the patient discontinued ALIS treatment. Although chest CT on day 461 revealed partial improvement after ALIS discontinuation, the patient’s symptoms persisted. Bronchoscopy performed on day 477 showed lymphocytic infiltration and enlarged foamy macrophages in TBLB specimens, with no evidence of eosinophilic infiltration or granuloma formation. Similarly, lipid-laden foamy macrophages were observed in BAL fluid. Subsequently, the patient was treated with prednisolone (30 mg/day), resulting in marked clinical and radiologic improvement within 1 month (Fig. 4 C). 3.5. Pathological Findings in Patients with DI-ILD Three patients in the DI-ILD group underwent TBLB and BAL (Table 2 ). Biopsy samples were obtained from regions of ground-glass opacity identified on HRCT. Specimens were collected from the right lower lobe in two patients and the right upper lobe in one. In all TBLB specimens, infiltration of enlarged foamy macrophages within alveolar epithelium was observed. Oil Red O staining performed on two TBLB sample (#3 and #7) confirmed the presence of lipid-laden foamy macrophages. No eosinophilic infiltration or granulomas were detected, and all samples tested negative in acid-fast staining. BAL fluid from two patients (#3 and #7) was positive for Oil Red O staining. BAL cytology from the DI-ILD cases demonstrated total cell counts of 170–680 cells/µL, with macrophages as the predominant cell type (47%–75%), followed by variable proportions of neutrophils (11%–34%) and lymphocytes (10%–22%). Eosinophils were rare (0%–2%). As a representative case, patient #3 underwent evaluation, with TBLB revealing enlarged foamy macrophages (Figs. 5 A, B). Consistently, BAL cytology demonstrated lipid-laden foamy macrophages on Oil Red O staining (Figs. 5 C, D). Table 2 Pathological Findings in Patients Diagnosed with DI-ILD Associated with ALIS Patient No. Biopsy Site Interval from Last ALIS Dose to Biopsy TBLB Findings BAL Fluid Findings #2 Right lower lobe 4 days Infiltration of large foamy macrophages in alveolar spaces (Oil Red O staining not performed). No eosinophilic infiltration or granuloma formation. Acid-fast staining is negative. Histiocytes and lymphocytes present (Oil Red O not performed). Total cells 680/µL; neutrophils 11%, lymphocytes 22%, macrophages 66%, eosinophils 1%. #3 Right upper lobe 33 days Infiltration of large foamy macrophages, positive for Oil Red O staining, in alveolar spaces. No eosinophilic infiltration or granuloma formation.Acid-fast staining is negative. Large foamy macrophages that are positive on Oil Red O staining. Total cells: 284/µL; neutrophils: 13%, lymphocytes: 10%, macrophages: 75%, eosinophils: 2%. #7 Right lower lobe 1 day Infiltration of large foamy macrophages that are positive for Oil Red O staining in alveolar spaces. No eosinophilic infiltration or granuloma formation. Acid-fast staining is negative. Large foamy macrophages that are positive on Oil Red O staining. Total cells: 170/µL; neutrophils: 34%, lymphocytes: 19%, macrophages: 47%, eosinophils: 0% 3.6. Pathological Findings in Patients Without DI-ILD Sputum culture conversion was not achieved during ALIS therapy; thus, SLR was performed in three patients who were not diagnosed with DI-ILD. In SLR specimens from all three non–DI-ILD patients, infiltration of enlarged foamy macrophages within the alveolar spaces was observed (Fig. 6 and Table 3 ). Oil Red O staining, performed in all three cases, was positive, confirming lipid-laden macrophages. A representative case from the non-DI-ILD group, including chest CT and pathological findings, is presented in Figs. 6 (A)–(C) and 7. The resected lung specimen was obtained 274 days after the ALIS initiation and 1 day after the final inhalation. Chest CT performed before the ALIS treatment showed a thick-walled cavity and centrilobular nodules in the right upper lobe (Fig. 7 A), which improved after 192 days of treatment (Fig. 7 B). During treatment, no new pulmonary infiltrates or ground-glass opacities suggestive of DI-ILD were observed. Due to persistent culture positivity, SLR was performed 274 days after ALIS initiation. Surgical lung resection was performed one day after the last ALIS inhalation. Histopathological examination revealed enlarged foamy macrophages in alveolar spaces. These macrophages were morphologically similar to those found in patients with DI-ILD and tested positive in Oil Red O staining. Pathology demonstrated similar findings of Oil Red O -positive enlarged foamy macrophages within the alveolar spaces in the other two patients—one who received ALIS for 52 days and the other for 302 days, each with a one-day interval after the last inhalation. Table 3 Pathological Findings in Surgical Resection Specimens from Patients Without ALIS-Associated DI-ILD Patient No. Resection Site Cumulative ALIS Treatment Duration/Interval from Last ALIS Dose to Surgery Pathological Findings #9 Right upper lobe 274 days/1 day Infiltration of large foamy macrophages, positive for Oil Red O staining, in alveolar spaces (Figs. 6 A–C). #10 Left lower lobe 52 days/1 day Infiltration of large foamy macrophages, positive for Oil Red O staining, in alveolar spaces (Figs. 6 D–F). #11 Right upper lobe 302 days/1 day Infiltration of large foamy macrophages, positive for Oil Red O staining, in alveolar spaces (Figs. 6 G–I) a Abbreviations : ALIS = amikacin liposomal inhalation suspension 4. Discussion This study investigated the pathological and clinical characteristics of ALIS-associated DI-ILD in patients with MAC-LD. Our findings indicate that lipid-laden foamy macrophages infiltrate the alveolar spaces of ALIS-treated patients, regardless of whether they develop DI-ILD. To the best of our knowledge, this is the first study to pathologically demonstrate the presence of lipoid-stain-positive foamy macrophages within the alveolar spaces of ALIS-treated patients. In this study, lipid-laden foamy macrophages were initially identified in SLR specimens from ALIS-treated patients without DI-ILD, representing the first pathological evidence that alveolar macrophages take up ALIS components in vivo . ALIS is a liposomal formulation of amikacin designed to enhance intracellular delivery to alveolar macrophages and improve bactericidal activity against MAC [ 11 ]. In murine models, alveolar macrophage concentrations of amikacin following ALIS inhalation were reportedly 274-fold higher than those following intravenous administration [ 11 ]. In our study, foamy, lipid-laden macrophages that infiltrate the alveolar spaces were observed in all patients who underwent pathological evaluation, thereby providing human evidence that alveolar macrophages internalize inhaled lipids. ALIS has persisted in sputum for over 72 h after inhalation [ 12 ]. From a pharmacokinetic perspective, inhaled liposomal antimicrobial formulations have provided slow intrapulmonary release of the drug, thereby enabling less frequent dosing while sustaining lung concentrations above the minimal inhibitory concentration for prolonged periods [ 13 ]. A gamma scintigraphy study in healthy volunteers demonstrated that inhaled liposomal amikacin achieved lung deposition of roughly one-third of the emitted dose and that approximately 60% and 38% of the initial lung burden remained at 24 and 48 h, respectively, indicating prolonged intrapulmonary retention [ 14 ]. Persistence of liposomal amikacin within human alveolar macrophages has not been previously confirmed; however, in patient #3, lipid-laden foamy macrophages were present in BAL fluid 30 days after ALIS discontinuation. This observation indicates that ALIS may persist within alveolar macrophages for a prolonged period and continue to exert local effects. ALIS-associated DI-ILD has been reported with patterns that resemble pulmonary alveolar proteinosis, organizing pneumonia, and hypersensitivity pneumonitis [ 3 , 8 , 9 ]. Pathology in all DI-ILD cases in our study demonstrated lipid-laden foamy macrophage characteristic of lipoid pneumonia; however, only small bronchoscopic specimens were available; thus, we could not definitively exclude hypersensitivity pneumonitis or organizing pneumonia as previously described [ 3 , 8 ]. The lipid component of ALIS includes cholesterol, an animal-derived lipid known to cause lipoid pneumonia following prolonged inhalation exposure [ 15 ]. In most DI-ILD cases, radiologic improvement on chest CT required > 1 month, and lipid-laden macrophages persisted up to 1 month after ALIS discontinuation in at least one patient. These findings are consistent with previous reports describing prolonged recovery period associated with exogenous lipoid pneumonia, which may range from 2 weeks to 8 months [ 15 ]. Accordingly, the differential diagnosis of ALIS-associated DI-ILD should include exogenous lipoid pneumonia. In our DI-ILD cohort, 63% of patients continued ALIS treatment with a reduced dosing frequency from daily to every other day. This observation supports the possibility that ALIS-associated DI-ILD may be prevented or mitigated by reducing exposure over a defined period. Such a pattern is biologically plausible and may occur in pneumonitis induced by allergic mechanisms (e.g., HP) or by cumulative lipid deposition, as in lipoid pneumonia [ 16 , 17 ]. Conversely, in CONVERT—with a 6-month observation window—the incidence of hypersensitivity pneumonitis was 3.1%, whereas allergic alveolitis that led to discontinuation occurred in 2.7% of patients in the prior-ALIS cohort in the 12-month extension study INS-312 [ 3 , 7 ]. In our series, five cases occurred by month 6, one between months 6 and 12, and two after month 12, indicating that some events appeared only with sustained exposure over time. Moreover, DI-ILD was reported in 14.7% of patients in the Japanese cohort of CONVERT [ 18 ], indicating that the incidence may vary in terms of observation window and population. Thus, DI-ILD may be preventable in some patients by reducing short-term exposure, whereas it can also arise with long-term exposure at a constant dose. Thus, careful attention to symptoms and chest imaging is warranted in both treatment phases. Notably, among the five patients diagnosed with DI-ILD who continued ALIS with every-other-day dosing after DI-ILD onset, three (60%) subsequently achieved sputum culture conversion. Treatment continuation with modified dosing rather than complete discontinuation may allow patients to maintain clinical benefits. In our study, serum biomarkers, including CRP, LDH and eosinophil count, were not significantly elevated in DI-ILD cases. However, KL-6 levels significantly increased at DI-ILD diagnosis compared with baseline. This finding is consistent with previous reports describing elevated KL-6 and CRP levels in patients with DI-ILD [ 8 , 19 ]. KL-6 is a high-molecular-weight glycoprotein that is predominantly expressed on type II alveolar and bronchiolar epithelial cells. Its serum concentration increases in response to alveolar epithelial injury and regeneration [ 20 , 21 ]. Increased KL-6 levels have been reported in various interstitial lung diseases, including idiopathic pulmonary fibrosis, connective tissue disease-associated ILD, and hypersensitivity pneumonitis, thereby reflecting disease activity and prognosis [ 20 , 22 ]. Further, KL-6 levels are frequently increased in patients with severe DI-ILD, including diffuse alveolar damage CT pattern or fibrotic ILD [ 22 , 23 ]. These findings, when considered alongside clinical findings, suggest that KL-6 is a potentially informative biomarker in patients with DI-ILD. However, these biomarkers lack specificity. Thus, clinicians should interpret KL-6 and other serum markers in conjunction with chest CT findings when evaluating patients presenting with fever, cough or dyspnoea during ALIS treatment. This study has some limitations. First, it was a single-centre, retrospective study with a small sample size. Second, the DI-ILD diagnosis was based on clinical judgement without established diagnostic criteria; thus, misclassification bias cannot be completely excluded. Finally, only TBLB and BAL specimens, which can be obtained by minimally invasive procedures, were available in patients with DI-ILD; therefore, a definitive histopathological diagnosis could not be established. SLR or transbronchial lung cryobiopsy may be required to obtain a more accurate pathological diagnosis even in patients with DI-ILD. Larger prospective studies are warranted to confirm these findings. Furthermore, standardised diagnostic and management guidelines for ALIS-associated DI-ILD are required. 5. Conclusion Lipid deposition within the lungs was observed irrespective of DI-ILD, despite no pathognomonic findings being identified for ALIS-associated DI-ILD. Even when DI-ILD occurred, some patients continued therapy by reducing the dosing frequency while preserving therapeutic efficacy. These findings highlight the importance of vigilant monitoring and individualised DI-ILD management to optimise outcomes in patients receiving ALIS. Abbreviations ALIS – amikacin liposomal inhalation suspension BAL – bronchoalveolar lavage BMI – body mass index CRP – C-reactive protein CT – computed tomography DI-ILD – drug-induced interstitial lung disease eGFR – estimated glomerular filtration rate FVC – forced vital capacity HRCT – high-resolution computed tomography ILD – interstitial lung disease KL-6 – Krebs von den Lungen-6 LDH – lactate dehydrogenase MAC – Mycobacterium avium complex MAC-LD – Mycobacterium avium complex lung disease SLR – surgical lung resection TBLB – transbronchial lung biopsy Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Toho University Medical Center Sakura Hospital (Approval number: S23058_S23006). The requirement for informed consent was waived owing to the retrospective nature of the study. Instead, the study details were publicly available on the institutional website, allowing potential participants to opt out. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding Not applicable. Author Contributions: CRediT HW and KI had the idea for and conceptualized the study. HW, KI, and YM had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. HW curated the data, performed the formal analysis, prepared the visualizations, and drafted the original version of the manuscript. NH, SK, AS, and YM contributed to data curation, investigation, and visualization. KI and YM supervised the study. 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Utility of KL-6/MUC1 in the clinical management of interstitial lung diseases. Respir Investig. 2012;50:3–13. He J, Zhang J, Ren X. Krebs von den lungen-6 as a clinical marker for hypersensitivity pneumonitis: A meta-analysis and bioinformatics analysis. Front Immunol. 2022;13:1041098. Ohnishi H, Circulating. KL-6 levels in patients with drug-induced interstitial lung disease. Thorax. 2003;58:872–5. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Mar, 2026 Read the published version in BMC Pulmonary Medicine → Version 1 posted Editorial decision: Revision requested 16 Jan, 2026 Reviews received at journal 13 Jan, 2026 Reviews received at journal 13 Jan, 2026 Reviewers agreed at journal 13 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviews received at journal 03 Jan, 2026 Reviewers agreed at journal 29 Dec, 2025 Reviewers invited by journal 08 Dec, 2025 Editor invited by journal 03 Dec, 2025 Editor assigned by journal 01 Dec, 2025 Submission checks completed at journal 01 Dec, 2025 First submitted to journal 28 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8234124","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":558475153,"identity":"ef3a9bc8-bbfa-49b5-8f0b-86b0e5a6b275","order_by":0,"name":"Hiroki 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1","display":"","copyAsset":false,"role":"figure","size":3182780,"visible":true,"origin":"","legend":"\u003cp\u003eStudy flow diagram\u003c/p\u003e\n\u003cp\u003eStudy flowchart showing patient selection and grouping into DI-ILD and non-DI-ILD cohorts.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e ALIS = amikacin liposomal inhalation suspension; MAC-LD = \u003cem\u003eMycobacterium avium\u003c/em\u003e complex lung disease; DI-ILD = drug-induced interstitial lung disease; TBLB = transbronchial lung biopsy; SLR = surgical lung resection\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8234124/v1/f384c30ca46cd3be8e704743.png"},{"id":97989139,"identity":"26643430-b98a-482c-a07d-12e4f8429145","added_by":"auto","created_at":"2025-12-11 14:23:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":709458,"visible":true,"origin":"","legend":"\u003cp\u003eSwimmer plot of patients diagnosed with DI-ILD associated with ALIS\u003c/p\u003e\n\u003cp\u003eEach horizontal bar represents a patient (#1–#8). Solid lines indicate the duration of daily ALIS administration. Red circles indicate the timing of DI-ILD diagnosis. Dotted lines represent the continuation of ALIS treatment at a reduced dosing frequency (every other day) after DI-ILD onset. Green diamonds indicate the timing of sputum culture conversion to negative. Grey triangles indicate the timing of bronchoscopy. Arrowheads denote patients still receiving treatment at the time of data cutoff. The average duration from ALIS initiation to DI-ILD diagnosis was 98.5 ± 159.8 days, and the mean time from DI-ILD diagnosis to radiologic improvement on chest CT was 43 ± 32 days. Five of eight patients (63%) were able to continue ALIS treatment with dosing frequency adjustment to every other day, and three patients achieved negative sputum conversion after reducing the dosing frequency.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e ALIS = amikacin liposomal inhalation suspension; CT = computed tomography; DI-ILD = drug-induced interstitial lung disease\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8234124/v1/c5de642756ea725e5f36f847.png"},{"id":98424172,"identity":"62236234-397f-4c4c-ac55-fbc49d26abd7","added_by":"auto","created_at":"2025-12-17 16:33:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1819427,"visible":true,"origin":"","legend":"\u003cp\u003eChange in serum CRP, KL-6 and LDH levels as well as eosinophil count in patients with DI-ILD associated with ALIS\u003c/p\u003e\n\u003cp\u003eChanges in serum CRP, KL-6 and LDH levels as well as eosinophil count between baseline and DI-ILD diagnosis. The KL-6 level significantly increased (\u003cem\u003eP\u003c/em\u003e = 0.0422).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e ALIS = amikacin liposomal inhalation suspension; DI-ILD = drug-induced interstitial lung disease; CRP = C-reactive protein; KL-6 = Krebs von den Lungen-6; LDH = lactate dehydrogenase\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8234124/v1/fe61d2f3596b495af4c319df.png"},{"id":97989143,"identity":"e757e273-065e-4035-8014-2e32a0a1c759","added_by":"auto","created_at":"2025-12-11 14:23:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4222031,"visible":true,"origin":"","legend":"\u003cp\u003eChest CT findings in a representative DI-ILD case (Patient #3)\u003c/p\u003e\n\u003cp\u003eSerial chest CT images of Patient #3 showing disease progression and response to therapy.\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp; Baseline CT scan performed before ALIS initiation.\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp; \u0026nbsp;High-resolution CT (HRCT) on the day of DI-ILD diagnosis, showing patchy ground-glass opacities with interlobular septal thickening.\u003c/p\u003e\n\u003cp\u003e(C)\u0026nbsp; \u0026nbsp;Complete resolution of ground-glass opacities after 1 month of corticosteroid therapy.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e CT = computed tomography; HRCT = high-resolution computed tomography; ALIS = amikacin liposomal inhalation suspension; DI-ILD = drug-induced interstitial lung disease\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8234124/v1/d9ff951a71789aa1fc258025.png"},{"id":97989162,"identity":"aaf629ee-8804-4dd9-8b8a-c80b2c6e7de7","added_by":"auto","created_at":"2025-12-11 14:23:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7778147,"visible":true,"origin":"","legend":"\u003cp\u003ePathological findings of DI-ILD case (Patient #3)\u003c/p\u003e\n\u003cp\u003ePathological findings from bronchoscopy performed 1 month after ALIS discontinuation in Patient #3.\u003c/p\u003e\n\u003cp\u003e(A, B) TBLB specimens show enlarged foamy macrophages (H\u0026amp;E staining; A: ×10, B: ×40). Arrows indicate lipid-laden foamy macrophages.\u003c/p\u003e\n\u003cp\u003e(C, D) BAL fluid demonstrating lipid-laden foamy macrophages on Oil Red O staining (C: ×10, D: ×40).\u003c/p\u003e\n\u003cp\u003eMagnifications in the figure legends indicate only the objective lens power. Scale bars: A = 100 µm; B = 20 µm; C = 100 µm; D = 20 µm.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e DI-ILD = drug-induced interstitial lung disease; TBLB = transbronchial lung biopsy; BAL = bronchoalveolar lavage; H\u0026amp;E = hematoxylin and eosin\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8234124/v1/0b5e4f0dfe3948976c08a131.png"},{"id":97989170,"identity":"8c3a16bb-dbbe-47ec-b5ea-9e77e4c583c6","added_by":"auto","created_at":"2025-12-11 14:23:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30668315,"visible":true,"origin":"","legend":"\u003cp\u003ePathological findings in SLR specimens from patients without ALIS-associated DI-ILD\u003c/p\u003e\n\u003cp\u003eSLR specimen from three patients without DI-ILD during ALIS treatment. All samples demonstrate alveolar infiltration of enlarged foamy macrophages. These cells are positive for Oil Red O staining.\u003c/p\u003e\n\u003cp\u003e(A–C) Patient #9. After 274 days of ALIS inhalation, surgery was performed one day following the last inhalation.\u003c/p\u003e\n\u003cp\u003e(A, B) Oil Red O staining demonstrating lipid-laden foamy macrophages in the alveolar spaces (A, ×10; B, ×40).\u003c/p\u003e\n\u003cp\u003e(C) H\u0026amp;E staining showing enlarged foamy macrophages (×40).\u003c/p\u003e\n\u003cp\u003e(D–F) Patient #10. After 52 days of ALIS inhalation, surgery was performed one day following the last inhalation.\u003c/p\u003e\n\u003cp\u003e(D, E) Oil Red O staining demonstrating lipid-laden foamy macrophages in the alveolar spaces (D, ×10; E, ×20).\u003c/p\u003e\n\u003cp\u003e(F) H\u0026amp;E staining showing enlarged foamy macrophages (×40).\u003c/p\u003e\n\u003cp\u003e(G–I) Patient #11. After 302 days of ALIS inhalation, surgery was performed one day after the last inhalation.\u003c/p\u003e\n\u003cp\u003e(G, H) H\u0026amp;E staining showing enlarged foamy macrophages in the alveolar spaces (G, ×4; H, ×10).\u003c/p\u003e\n\u003cp\u003e(I) Oil Red O staining demonstrating lipid-laden foamy macrophages (×40).\u003c/p\u003e\n\u003cp\u003eMagnifications in the figure legends indicate only the objective lens power. Scale bars: A, D, H = 100 µm; B, C, F, I = 20 µm; E = 50 µm; G = 200 µm.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAbbreviations:\u003c/strong\u003e ALIS = amikacin liposomal inhalation suspension; DI-ILD = drug-induced interstitial lung disease; H\u0026amp;E = hematoxylin and eosin; SLR = surgical lung resection\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8234124/v1/01cd8bd72372d8a38c5827f5.png"},{"id":98424609,"identity":"c1da17f7-22f5-4100-9e86-fae256d46446","added_by":"auto","created_at":"2025-12-17 16:33:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3119272,"visible":true,"origin":"","legend":"\u003cp\u003eChest CT findings in patients without DI-ILD treated with ALIS\u003c/p\u003e\n\u003cp\u003eSerial chest CT images from a patient who underwent surgical lung resection during ALIS treatment.\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp; CT scan performed before ALIS initiation showing a thick-walled cavity and centrilobular nodules in the right upper lobe.\u003c/p\u003e\n\u003cp\u003e(B)\u0026nbsp; \u0026nbsp;Preoperative CT scan after ALIS initiation showing thinning of the cavity wall, improvement of centrilobular nodules, and no apparent findings suggestive of DI-ILD.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eAbbreviations: \u003c/strong\u003eCT = computed tomography; ALIS = amikacin liposomal inhalation suspension; DI-ILD = drug-induced interstitial lung disease\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8234124/v1/2c530049ea52cba9e269eb0f.png"},{"id":104739613,"identity":"89395d9f-24e6-4ea0-a8cd-57c4a0b3f932","added_by":"auto","created_at":"2026-03-16 16:10:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":51312732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8234124/v1/f2be5ddb-bcbf-4a9d-9cbe-c80370cdd00d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lipid Accumulation and Drug-Induced Interstitial Lung Disease Associated with Amikacin Liposomal Inhalation Suspension in Patients with Mycobacterium avium Complex Lung Disease: A Singlecenter Retrospective Cohort Study","fulltext":[{"header":"1. Background","content":"\u003cp\u003e\u003cem\u003eMycobacterium avium\u003c/em\u003e complex lung disease (MAC-LD) is a chronic pulmonary infection caused by nontuberculous mycobacteria, predominantly \u003cem\u003eMycobacterium avium\u003c/em\u003e complex (MAC) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Japan, the clinical burden of MAC-LD has been increasing since 2010, surpassing the incidence of new tuberculosis cases in 2018 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite long-term multidrug regimens, MAC-LD management remains challenging owing to frequent relapse and treatment refractoriness [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to international guidelines, the standard treatment regimen includes macrolides, ethambutol and rifamycin, with aminoglycosides added for patients with severe or refractory disease [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Injectable aminoglycosides, such as amikacin, streptomycin and kanamycin, have been used traditionally; however, in 2020, amikacin liposomal inhalation suspension (ALIS) was approved by the US Food and Drug Administration as an alternative [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eALIS is indicated for patients with refractory or relapsed MAC-LD; it has demonstrated efficacy when combined with guideline-based therapy, achieving sputum culture conversion in approximately 29% of refractory cases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Compared with intravenous aminoglycosides, ALIS produces lower serum concentrations, potentially minimising the risk of ototoxicity, nephrotoxicity and vestibular toxicity while preserving antimicrobial efficacy [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite these advantages, ALIS has been associated with specific adverse events such as hoarseness and drug-induced interstitial lung disease (DI-ILD) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The CONVERT study, an international phase 3 open-label trial, reported hypersensitivity pneumonitis in 3.1% of patients receiving ALIS, which led to treatment discontinuation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition, several case reports have described the occurrence of interstitial pneumonia in ALIS-treated patients [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the pathophysiological mechanisms underlying ALIS-associated DI-ILD remain poorly understood.\u003c/p\u003e\u003cp\u003eThis study aimed to investigate the aetiology of ALIS-associated DI-ILD by evaluating the clinical characteristics, laboratory results, high-resolution chest computed tomography (HRCT) findings and pathological findings of patients with MAC-LD receiving ALIS treatment.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Study Design\u003c/h2\u003e\u003cp\u003eThis single-centre retrospective cohort study was conducted at Toho University Medical Center, Sakura Hospital. The study protocol was approved by the hospital\u0026rsquo;s institutional ethics committee (approval number: S23058_S23006). The requirement for informed consent was waived owing to the retrospective nature of the study. Instead, the study details were publicly available on the institutional website, allowing potential participants to opt out.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Patient Population\u003c/h2\u003e\u003cp\u003e The study included patients diagnosed with MAC-LD based on the 2020 ATS/ERS/ESCMID/IDSA clinical practice guidelines who received ALIS treatment between September 2021 and October 2024. Patients with an observation period of at least 6 months after the ALIS initiation were considered eligible.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Data Collection\u003c/h2\u003e\u003cp\u003ePatient data, including age, sex, body mass index (BMI), negative sputum conversion status, treatment duration, and DI-ILD development, were extracted from electronic medical records. Negative sputum conversion was defined as three consecutive negative sputum cultures that are collected at intervals of at least one month. The date of sputum culture conversion was assigned as the date on which the first of these three negative samples was submitted. The conversion date, for patients in whom sputum production resolved with treatment and samples could no longer be submitted, was defined as the first outpatient visit at which sputum could not be provided. Furthermore, spirometry as well as laboratory, HRCT and pathological findings from transbronchial lung biopsy (TBLB), bronchoalveolar lavage (BAL) and surgical lung resection for treatment (SLR) were reviewed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. ALIS Administration\u003c/h2\u003e\u003cp\u003eAll patients received 590 mg of ALIS (Arikayce; Insmed Inc., Bridgewater, NJ) administered using the eFlow\u0026reg; rapid nebuliser system (Lamira; PARI Pharma GmbH, Munich, Germany). All patients were trained on proper inhalation techniques during a 3\u0026ndash;7-day hospitalisation period or at least three outpatient visits. Inhalation adherence and technique were evaluated monthly by respiratory specialists and trained nurses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Diagnostic Criteria for ALIS-Associated DI-ILD\u003c/h2\u003e\u003cp\u003eDue to the absence of established diagnostic criteria for ALIS-associated DI-ILD, the diagnosis in this study was based on the following conditions: (1) appearance of new bilateral consolidations or ground-glass opacities on chest computed tomography (CT) following ALIS initiation, which improved with dose reduction or treatment discontinuation; (2) presence of at least one symptom, such as fever, cough or dyspnoea; (3) negative PCR test for severe acute respiratory syndrome coronavirus 2; (4) no newly introduced medications within 1 month before symptom onset; (5) no history of autoimmune disease or HIV/AIDS; (6) no use of corticosteroids or other immunosuppressive agents; and (7) other causes, such as bacterial pneumonia, viral infections (e.g., influenza), heart failure, and progression of MAC-LD, were excluded based on clinical course, microbiological tests, and imaging findings. The date of DI-ILD diagnosis was defined as the time when abnormal findings were first identified on chest CT. Two thoracic radiologists and two pulmonologists independently reviewed CT scans to reach a final diagnosis of DI-ILD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Laboratory Evaluation\u003c/h2\u003e\u003cp\u003eAt ALIS initiation and DI-ILD onset, the levels of serum biomarkers associated with interstitial lung disease\u0026mdash;C-reactive protein (CRP), lactate dehydrogenase (LDH), Krebs von den Lungen-6 (KL-6) and eosinophil count\u0026mdash;were measured. Furthermore, the estimated glomerular filtration rate (eGFR) was evaluated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Chest CT Assessment\u003c/h2\u003e\u003cp\u003eHRCT was performed before and after ALIS initiation to assess pulmonary involvement. Baseline disease severity was scored using a previously validated CT scoring system [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Pathological Assessment\u003c/h2\u003e\u003cp\u003eLung tissue from TBLB was fixed in 10% neutral buffered formalin, and SLR specimens were fixed using bronchial perfusion and immersion methods. Tissue processing involved sequential infiltration with 10%, 15% and 30% sucrose solutions at 4\u0026deg;C, followed by embedding in Tissue-Tek\u0026reg; optimal cutting temperature compound and then freezing in liquid nitrogen. Tissue sections were cut at 5\u0026ndash;6-\u0026micro;m thickness for hematoxylin and eosin and Oil Red O staining when foamy macrophages were observed. BAL fluid samples were centrifuged, with the smears similarly fixed and stained.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e\u003cp\u003eCategorical variables were expressed as numbers and percentages, whereas continuous variables were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Categorical variables were compared using the chi-squared or Fisher\u0026rsquo;s exact test, as appropriate, whereas continuous variables were compared using the Mann\u0026ndash;Whitney U test. Paired \u003cem\u003et\u003c/em\u003e-tests were used to assess changes in biomarkers between baseline and the time of DI-ILD diagnosis. Analyses were conducted using the SPSS software (version 25; IBM Corp., Armonk, NY, USA), and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered to indicate statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Study Population\u003c/h2\u003e\u003cp\u003eThe study flow is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A total of 32 patients with MAC-LD who received ALIS treatment between September 2021 and October 2024 were initially enrolled in the study. Of them, seven were excluded as they had an observation period of \u0026lt;\u0026thinsp;6 months after ALIS initiation. Consequently, 25 patients were included in the final analysis, of whom 8 (32%) developed DI-ILD (DI-ILD group) and 17 (68%) did not (non-DI-ILD group). During ALIS treatment, three patients in the DI-ILD group underwent TBLB and BAL, whereas three in the non-DI-ILD group underwent SLR.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe patients\u0026rsquo; baseline characteristics are summarised in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The mean age of the cohort was 74\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2 years. Of the included patients, 16% were male and 84% were female. The mean BMI was 18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 kg/m\u0026sup2;. No statistically significant differences were observed between the groups in pulmonary function parameters, sputum culture conversion rate or chest CT scoring. However, the DI-ILD group had a significantly lower eGFR than the non-DI-ILD group (62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.8 vs. 78.9\u0026thinsp;\u0026plusmn;\u0026thinsp;13.3 mL/min/1.73 m\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBaseline Characteristics of the Study Population\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAll Patients (n\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDI-ILD Group (n\u0026thinsp;=\u0026thinsp;8)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNon-DI-ILD Group (n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (years), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74\u0026thinsp;\u0026plusmn;\u0026thinsp;7.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e73\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e74\u0026thinsp;\u0026plusmn;\u0026thinsp;8.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.608\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex, n (%), F/M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21/4 (84/16)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 /0(100/0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13/4 (76/24)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.269\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBMI (kg/m\u0026sup2;), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.932\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNegative sputum conversion, n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13 (52)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5 (63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8 (47)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.673\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment duration (days), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e404\u0026thinsp;\u0026plusmn;\u0026thinsp;257\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e285\u0026thinsp;\u0026plusmn;\u0026thinsp;158\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e460\u0026thinsp;\u0026plusmn;\u0026thinsp;278\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.114\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%FEV\u003csub\u003e1\u003c/sub\u003e, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e92.6\u0026thinsp;\u0026plusmn;\u0026thinsp;25.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.5\u0026thinsp;\u0026plusmn;\u0026thinsp;26.8\u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.052\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e%FVC, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e86.5\u0026thinsp;\u0026plusmn;\u0026thinsp;23.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e77.2\u0026thinsp;\u0026plusmn;\u0026thinsp;15.4*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e91.2\u0026thinsp;\u0026plusmn;\u0026thinsp;25.3\u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.237\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCT score, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.880\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCRP (mg/dL), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.708\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLDH (U/L), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e195\u0026thinsp;\u0026plusmn;\u0026thinsp;43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e185\u0026thinsp;\u0026plusmn;\u0026thinsp;19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e201\u0026thinsp;\u0026plusmn;\u0026thinsp;51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.413\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKL-6 (U/mL), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e494\u0026thinsp;\u0026plusmn;\u0026thinsp;240\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e472\u0026thinsp;\u0026plusmn;\u0026thinsp;247\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e507\u0026thinsp;\u0026plusmn;\u0026thinsp;245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.746\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eeGFR (mL/min/1.73 m\u0026sup2;), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e73.2\u0026thinsp;\u0026plusmn;\u0026thinsp;15.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e78.9\u0026thinsp;\u0026plusmn;\u0026thinsp;13.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.010\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEosinophil count (/\u0026micro;L), mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e171\u0026thinsp;\u0026plusmn;\u0026thinsp;168\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e189\u0026thinsp;\u0026plusmn;\u0026thinsp;178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e163\u0026thinsp;\u0026plusmn;\u0026thinsp;169\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.839\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003e * (n\u0026thinsp;=\u0026thinsp;6), \u0026dagger; (n\u0026thinsp;=\u0026thinsp;12)\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e\u003cb\u003eb\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eAbbreviations\u003c/b\u003e: SD = standard deviation; BMI\u0026thinsp;=\u0026thinsp;body mass index; CRP\u0026thinsp;=\u0026thinsp;C-reactive protein; DI-ILD\u0026thinsp;=\u0026thinsp;drug-induced interstitial lung disease; eGFR\u0026thinsp;=\u0026thinsp;estimated glomerular filtration rate; FEV\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;forced expiratory volume in 1 second; FVC\u0026thinsp;=\u0026thinsp;forced vital capacity; KL-6\u0026thinsp;=\u0026thinsp;Krebs von den Lungen-6; LDH\u0026thinsp;=\u0026thinsp;lactate dehydrogenase\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Clinical Course of DI-ILD\u003c/h2\u003e\u003cp\u003eThe clinical courses of the eight patients diagnosed with ALIS-associated DI-ILD are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The mean duration from ALIS initiation to DI-ILD diagnosis was 98.5\u0026thinsp;\u0026plusmn;\u0026thinsp;159.8 days. Following DI-ILD onset, seven of the eight patients exhibited radiologic and symptomatic improvement after either treatment discontinuation or adjustment to alternate-day dosing. The mean duration from DI-ILD diagnosis to radiologic improvement on chest CT was 43\u0026thinsp;\u0026plusmn;\u0026thinsp;32 days. Remaining one patient required oral corticosteroid therapy owing to persistent symptoms. Notably, of the 8 patients, 5 (63%) continued ALIS by reducing the dosing frequency to every-other-day inhalation, whereas 3 achieved negative sputum conversion after reducing the dosing frequency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Laboratory Findings\u003c/h2\u003e\u003cp\u003eChanges in serum CRP, KL-6 and LDH levels as well as eosinophil count from baseline to DI-ILD diagnosis are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. No significant changes were observed in CRP, LDH or eosinophil count between the two time points. However, KL-6 levels significantly increased at DI-ILD diagnosis compared with that at baseline (407\u0026thinsp;\u0026plusmn;\u0026thinsp;231 vs. 434\u0026thinsp;\u0026plusmn;\u0026thinsp;250 U/mL; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0422).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Representative DI-ILD Case Findings\u003c/h2\u003e\u003cp\u003eRepresentative chest CT and pathological findings from Patient #3\u0026mdash;the only case requiring corticosteroid therapy\u0026mdash;are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A baseline CT scan performed before the ALIS initiation revealed no evidence of DI-ILD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The patient experienced cough and dyspnoea on day 444 after ALIS initiation. HRCT showed ground-glass opacities with interlobular septal thickening, raising suspicion for ALIS-associated DI-ILD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB); hence, the patient discontinued ALIS treatment. Although chest CT on day 461 revealed partial improvement after ALIS discontinuation, the patient\u0026rsquo;s symptoms persisted. Bronchoscopy performed on day 477 showed lymphocytic infiltration and enlarged foamy macrophages in TBLB specimens, with no evidence of eosinophilic infiltration or granuloma formation. Similarly, lipid-laden foamy macrophages were observed in BAL fluid. Subsequently, the patient was treated with prednisolone (30 mg/day), resulting in marked clinical and radiologic improvement within 1 month (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Pathological Findings in Patients with DI-ILD\u003c/h2\u003e\u003cp\u003eThree patients in the DI-ILD group underwent TBLB and BAL (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Biopsy samples were obtained from regions of ground-glass opacity identified on HRCT. Specimens were collected from the right lower lobe in two patients and the right upper lobe in one. In all TBLB specimens, infiltration of enlarged foamy macrophages within alveolar epithelium was observed. Oil Red O staining performed on two TBLB sample (#3 and #7) confirmed the presence of lipid-laden foamy macrophages. No eosinophilic infiltration or granulomas were detected, and all samples tested negative in acid-fast staining. BAL fluid from two patients (#3 and #7) was positive for Oil Red O staining. BAL cytology from the DI-ILD cases demonstrated total cell counts of 170\u0026ndash;680 cells/\u0026micro;L, with macrophages as the predominant cell type (47%\u0026ndash;75%), followed by variable proportions of neutrophils (11%\u0026ndash;34%) and lymphocytes (10%\u0026ndash;22%). Eosinophils were rare (0%\u0026ndash;2%). As a representative case, patient #3 underwent evaluation, with TBLB revealing enlarged foamy macrophages (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Consistently, BAL cytology demonstrated lipid-laden foamy macrophages on \u003cem\u003eOil Red O\u003c/em\u003e staining (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePathological Findings in Patients Diagnosed with DI-ILD Associated with ALIS\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatient No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBiopsy Site\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInterval from Last ALIS Dose to Biopsy\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTBLB Findings\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBAL Fluid Findings\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e#2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRight lower lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInfiltration of large foamy macrophages in alveolar spaces (Oil Red O staining not performed). No eosinophilic infiltration or granuloma formation. Acid-fast staining is negative.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHistiocytes and lymphocytes present (Oil Red O not performed). Total cells 680/\u0026micro;L; neutrophils 11%, lymphocytes 22%, macrophages 66%, eosinophils 1%.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e#3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRight upper lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33 days\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInfiltration of large foamy macrophages, positive for Oil Red O staining, in alveolar spaces. No eosinophilic infiltration or granuloma formation.Acid-fast staining is negative.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLarge foamy macrophages that are positive on Oil Red O staining. Total cells: 284/\u0026micro;L; neutrophils: 13%, lymphocytes: 10%, macrophages: 75%, eosinophils: 2%.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e#7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRight lower lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInfiltration of large foamy macrophages that are positive for Oil Red O staining in alveolar spaces. No eosinophilic infiltration or granuloma formation. Acid-fast staining is negative.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLarge foamy macrophages that are positive on Oil Red O staining. Total cells: 170/\u0026micro;L; neutrophils: 34%, lymphocytes: 19%, macrophages: 47%, eosinophils: 0%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Pathological Findings in Patients Without DI-ILD\u003c/h2\u003e\u003cp\u003eSputum culture conversion was not achieved during ALIS therapy; thus, SLR was performed in three patients who were not diagnosed with DI-ILD. In SLR specimens from all three non\u0026ndash;DI-ILD patients, infiltration of enlarged foamy macrophages within the alveolar spaces was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Oil Red O staining, performed in all three cases, was positive, confirming lipid-laden macrophages. A representative case from the non-DI-ILD group, including chest CT and pathological findings, is presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (A)\u0026ndash;(C) and 7. The resected lung specimen was obtained 274 days after the ALIS initiation and 1 day after the final inhalation. Chest CT performed before the ALIS treatment showed a thick-walled cavity and centrilobular nodules in the right upper lobe (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), which improved after 192 days of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). During treatment, no new pulmonary infiltrates or ground-glass opacities suggestive of DI-ILD were observed. Due to persistent culture positivity, SLR was performed 274 days after ALIS initiation. Surgical lung resection was performed one day after the last ALIS inhalation. Histopathological examination revealed enlarged foamy macrophages in alveolar spaces. These macrophages were morphologically similar to those found in patients with DI-ILD and tested positive in Oil Red O staining. Pathology demonstrated similar findings of \u003cem\u003eOil Red O\u003c/em\u003e-positive enlarged foamy macrophages within the alveolar spaces in the other two patients\u0026mdash;one who received ALIS for 52 days and the other for 302 days, each with a one-day interval after the last inhalation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePathological Findings in Surgical Resection Specimens from Patients Without ALIS-Associated DI-ILD\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePatient No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResection Site\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCumulative ALIS Treatment Duration/Interval from Last ALIS Dose to Surgery\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePathological Findings\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e#9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRight upper lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e274 days/1 day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInfiltration of large foamy macrophages, positive for Oil Red O staining, in alveolar spaces (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e#10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLeft lower lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e52 days/1 day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInfiltration of large foamy macrophages, positive for Oil Red O staining, in alveolar spaces (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026ndash;F).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e#11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRight upper lobe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e302 days/1 day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInfiltration of large foamy macrophages, positive for Oil Red O staining, in alveolar spaces (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u0026ndash;I)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eAbbreviations\u003c/b\u003e: ALIS\u0026thinsp;=\u0026thinsp;amikacin liposomal inhalation suspension\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study investigated the pathological and clinical characteristics of ALIS-associated DI-ILD in patients with MAC-LD. Our findings indicate that lipid-laden foamy macrophages infiltrate the alveolar spaces of ALIS-treated patients, regardless of whether they develop DI-ILD. To the best of our knowledge, this is the first study to pathologically demonstrate the presence of lipoid-stain-positive foamy macrophages within the alveolar spaces of ALIS-treated patients.\u003c/p\u003e\u003cp\u003eIn this study, lipid-laden foamy macrophages were initially identified in SLR specimens from ALIS-treated patients without DI-ILD, representing the first pathological evidence that alveolar macrophages take up ALIS components \u003cem\u003ein vivo\u003c/em\u003e. ALIS is a liposomal formulation of amikacin designed to enhance intracellular delivery to alveolar macrophages and improve bactericidal activity against MAC [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In murine models, alveolar macrophage concentrations of amikacin following ALIS inhalation were reportedly 274-fold higher than those following intravenous administration [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In our study, foamy, lipid-laden macrophages that infiltrate the alveolar spaces were observed in all patients who underwent pathological evaluation, thereby providing human evidence that alveolar macrophages internalize inhaled lipids. ALIS has persisted in sputum for over 72 h after inhalation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. From a pharmacokinetic perspective, inhaled liposomal antimicrobial formulations have provided slow intrapulmonary release of the drug, thereby enabling less frequent dosing while sustaining lung concentrations above the minimal inhibitory concentration for prolonged periods [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A gamma scintigraphy study in healthy volunteers demonstrated that inhaled liposomal amikacin achieved lung deposition of roughly one-third of the emitted dose and that approximately 60% and 38% of the initial lung burden remained at 24 and 48 h, respectively, indicating prolonged intrapulmonary retention [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Persistence of liposomal amikacin within human alveolar macrophages has not been previously confirmed; however, in patient #3, lipid-laden foamy macrophages were present in BAL fluid 30 days after ALIS discontinuation. This observation indicates that ALIS may persist within alveolar macrophages for a prolonged period and continue to exert local effects.\u003c/p\u003e\u003cp\u003eALIS-associated DI-ILD has been reported with patterns that resemble pulmonary alveolar proteinosis, organizing pneumonia, and hypersensitivity pneumonitis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Pathology in all DI-ILD cases in our study demonstrated lipid-laden foamy macrophage characteristic of lipoid pneumonia; however, only small bronchoscopic specimens were available; thus, we could not definitively exclude hypersensitivity pneumonitis or organizing pneumonia as previously described [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The lipid component of ALIS includes cholesterol, an animal-derived lipid known to cause lipoid pneumonia following prolonged inhalation exposure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In most DI-ILD cases, radiologic improvement on chest CT required\u0026thinsp;\u0026gt;\u0026thinsp;1 month, and lipid-laden macrophages persisted up to 1 month after ALIS discontinuation in at least one patient. These findings are consistent with previous reports describing prolonged recovery period associated with exogenous lipoid pneumonia, which may range from 2 weeks to 8 months [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Accordingly, the differential diagnosis of ALIS-associated DI-ILD should include exogenous lipoid pneumonia.\u003c/p\u003e\u003cp\u003eIn our DI-ILD cohort, 63% of patients continued ALIS treatment with a reduced dosing frequency from daily to every other day. This observation supports the possibility that ALIS-associated DI-ILD may be prevented or mitigated by reducing exposure over a defined period. Such a pattern is biologically plausible and may occur in pneumonitis induced by allergic mechanisms (e.g., HP) or by cumulative lipid deposition, as in lipoid pneumonia [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Conversely, in CONVERT\u0026mdash;with a 6-month observation window\u0026mdash;the incidence of hypersensitivity pneumonitis was 3.1%, whereas allergic alveolitis that led to discontinuation occurred in 2.7% of patients in the prior-ALIS cohort in the 12-month extension study INS-312 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In our series, five cases occurred by month 6, one between months 6 and 12, and two after month 12, indicating that some events appeared only with sustained exposure over time. Moreover, DI-ILD was reported in 14.7% of patients in the Japanese cohort of CONVERT [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], indicating that the incidence may vary in terms of observation window and population. Thus, DI-ILD may be preventable in some patients by reducing short-term exposure, whereas it can also arise with long-term exposure at a constant dose. Thus, careful attention to symptoms and chest imaging is warranted in both treatment phases. Notably, among the five patients diagnosed with DI-ILD who continued ALIS with every-other-day dosing after DI-ILD onset, three (60%) subsequently achieved sputum culture conversion. Treatment continuation with modified dosing rather than complete discontinuation may allow patients to maintain clinical benefits.\u003c/p\u003e\u003cp\u003eIn our study, serum biomarkers, including CRP, LDH and eosinophil count, were not significantly elevated in DI-ILD cases. However, KL-6 levels significantly increased at DI-ILD diagnosis compared with baseline. This finding is consistent with previous reports describing elevated KL-6 and CRP levels in patients with DI-ILD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. KL-6 is a high-molecular-weight glycoprotein that is predominantly expressed on type II alveolar and bronchiolar epithelial cells. Its serum concentration increases in response to alveolar epithelial injury and regeneration [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Increased KL-6 levels have been reported in various interstitial lung diseases, including idiopathic pulmonary fibrosis, connective tissue disease-associated ILD, and hypersensitivity pneumonitis, thereby reflecting disease activity and prognosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Further, KL-6 levels are frequently increased in patients with severe DI-ILD, including diffuse alveolar damage CT pattern or fibrotic ILD [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These findings, when considered alongside clinical findings, suggest that KL-6 is a potentially informative biomarker in patients with DI-ILD. However, these biomarkers lack specificity. Thus, clinicians should interpret KL-6 and other serum markers in conjunction with chest CT findings when evaluating patients presenting with fever, cough or dyspnoea during ALIS treatment.\u003c/p\u003e\u003cp\u003eThis study has some limitations. First, it was a single-centre, retrospective study with a small sample size. Second, the DI-ILD diagnosis was based on clinical judgement without established diagnostic criteria; thus, misclassification bias cannot be completely excluded. Finally, only TBLB and BAL specimens, which can be obtained by minimally invasive procedures, were available in patients with DI-ILD; therefore, a definitive histopathological diagnosis could not be established. SLR or transbronchial lung cryobiopsy may be required to obtain a more accurate pathological diagnosis even in patients with DI-ILD. Larger prospective studies are warranted to confirm these findings. Furthermore, standardised diagnostic and management guidelines for ALIS-associated DI-ILD are required.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eLipid deposition within the lungs was observed irrespective of DI-ILD, despite no pathognomonic findings being identified for ALIS-associated DI-ILD. Even when DI-ILD occurred, some patients continued therapy by reducing the dosing frequency while preserving therapeutic efficacy. These findings highlight the importance of vigilant monitoring and individualised DI-ILD management to optimise outcomes in patients receiving ALIS.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALIS – amikacin liposomal inhalation suspension\u003c/p\u003e\n\u003cp\u003eBAL – bronchoalveolar lavage\u003c/p\u003e\n\u003cp\u003eBMI – body mass index\u003c/p\u003e\n\u003cp\u003eCRP – C-reactive protein\u003c/p\u003e\n\u003cp\u003eCT – computed tomography\u003c/p\u003e\n\u003cp\u003eDI-ILD – drug-induced interstitial lung disease\u003c/p\u003e\n\u003cp\u003eeGFR – estimated glomerular filtration rate\u003c/p\u003e\n\u003cp\u003eFVC – forced vital capacity\u003c/p\u003e\n\u003cp\u003eHRCT – high-resolution computed tomography\u003c/p\u003e\n\u003cp\u003eILD – interstitial lung disease\u003c/p\u003e\n\u003cp\u003eKL-6 – Krebs von den Lungen-6\u003c/p\u003e\n\u003cp\u003eLDH – lactate dehydrogenase\u003c/p\u003e\n\u003cp\u003eMAC – Mycobacterium avium complex\u003c/p\u003e\n\u003cp\u003eMAC-LD – Mycobacterium avium complex lung disease\u003c/p\u003e\n\u003cp\u003eSLR – surgical lung resection\u003c/p\u003e\n\u003cp\u003eTBLB – transbronchial lung biopsy\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eEthics approval and consent to participate\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Toho University Medical Center Sakura Hospital (Approval number: S23058_S23006). The requirement for informed consent was waived owing to the retrospective nature of the study. Instead, the study details were publicly available on the institutional website, allowing potential participants to opt out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eConsent for publication\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAvailability of data and materials\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCompeting interests\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthor Contributions: CRediT\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHW and KI had the idea for and conceptualized the study. HW, KI, and YM had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. HW curated the data, performed the formal analysis, prepared the visualizations, and drafted the original version of the manuscript. NH, SK, AS, and YM contributed to data curation, investigation, and visualization. KI and YM supervised the study. All authors contributed to writing – review and editing, critically revised the manuscript for important intellectual content, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the medical staff of the Department of Respiratory Medicine, Toho University Medical Center Sakura Hospital, for their invaluable support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDaley CL, Iaccarino JM, Lange C, Cambau E, Wallace RJ, Andrejak C, et al. Treatment of nontuberculous mycobacterial pulmonary disease: an official ATS/ERS/ESCMID/IDSA clinical practice guideline. Eur Respir J. 2020;56:2000535.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIto M, Furuuchi K, Fujiwara K, Kodama T, Tanaka Y, Yoshiyama T, et al. Epidemiological trends and clinical relevance of nontuberculous mycobacterial pulmonary disease in a referral hospital in Japan, 2017\u0026ndash;2021. Respir Investig. 2017;62:1064\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGriffith DE, Eagle G, Thomson R, Aksamit TR, Hasegawa N, Morimoto K, et al. Amikacin Liposome Inhalation Suspension for Treatment-Refractory Lung Disease Caused by \u003cem\u003eMycobacterium avium\u003c/em\u003e Complex (CONVERT). A Prospective, Open-Label, Randomized Study. Am J Respir Crit Care Med. 2018;198:1559\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim SM, Chong YP, Lee HJ, Shim TS, Jo K-W. Comparison of Treatment Outcomes of Cavitary Mycobacterium avium Complex Pulmonary Disease with Streptomycin or Amikacin Use. Microbiol Spectr. 2023;11:474122.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorita A, Namkoong H, Yagi K, Asakura T, Hosoya M, Tanaka H, et al. Early-Phase Adverse Effects and Management of Liposomal Amikacin Inhalation for Refractory Mycobacterium avium Complex Lung Disease in Real-World Settings. Infect Drug Resist. 2022;15:4001\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGriffith DE, Thomson R, Flume PA, Aksamit TR, Field SK, Addrizzo-Harris DJ, et al. Amikacin Liposome Inhalation Suspension for Refractory Mycobacterium avium Complex Lung Disease. Chest. 2021;160:831\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWinthrop KL, Flume PA, Thomson R, Mange KC, Yuen DW, Ciesielska M, et al. Amikacin Liposome Inhalation Suspension for \u003cem\u003eMycobacterium avium\u003c/em\u003e Complex Lung Disease: A 12-Month Open-Label Extension Clinical Trial. Ann Am Thorac Soc. 2021;18:1147\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakao D, Takeda K, Takazono T, Ozasa M, Ito Y, Ashizawa N, et al. A case of drug-induced organizing pneumonia caused by amikacin liposome inhalation suspension. J Infect Chemother. 2023;29:806\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHashimoto K, Nii T, Sumitani H, Yokoyama M, Miyamoto S, Mihashi Y, et al. Diagnosis and Management of Drug-Induced Interstitial Lung Disease Associated with Amikacin Liposome Inhalation Suspension in Refractory Mycobacterium Avium Complex Pulmonary Disease: A Case Report. Infect Drug Resist. 2023;16:6629\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee G, Lee KS, Moon JW, Koh W-J, Jeong B-H, Jeong YJ, et al. Nodular Bronchiectatic \u003cem\u003eMycobacterium avium\u003c/em\u003e Complex Pulmonary Disease. Natural Course on Serial Computed Tomographic Scans. Ann Am Thorac Soc. 2013;10:299\u0026ndash;306.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang J, Leifer F, Rose S, Chun DY, Thaisz J, Herr T, et al. Amikacin Liposome Inhalation Suspension (ALIS) Penetrates Non-tuberculous Mycobacterial Biofilms and Enhances Amikacin Uptake Into Macrophages. Front Microbiol. 2018;9:915.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRubino CM, Onufrak NJ, van Ingen J, Griffith DE, Bhavnani SM, Yuen DW, et al. Population Pharmacokinetic Evaluation of Amikacin Liposome Inhalation Suspension in Patients with Treatment-Refractory Nontuberculous Mycobacterial Lung Disease. Eur J Drug Metab Pharmacokinet. 2021;46:277\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBassetti M, Vena A, Russo A, Peghin M. Inhaled Liposomal Antimicrobial Delivery in Lung Infections. Drugs. 2020;80:1309\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeers J, Metzheiser B, Taylor G, Warren S, Meers P, Perkins WR. A Gamma Scintigraphy Study to Investigate Lung Deposition and Clearance of Inhaled Amikacin-Loaded Liposomes in Healthy Male Volunteers. J Aerosol Med Pulm Drug Deliv. 2009;22:131\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBetancourt SL, Martinez-Jimenez S, Rossi SE, Truong MT, Carrillo J, Erasmus JJ. Lipoid Pneumonia: Spectrum of Clinical and Radiologic Manifestations. Am J Roentgenol. 2010;194:103\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTomioka H, Miyazaki Y, Inoue Y, Egashira R, Kawamura T, Sano H, et al. Japanese clinical practice guide 2022 for hypersensitivity pneumonitis. Respir Investig. 2024;62:16\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarchiori E, Zanetti G, Mano CM, Hochhegger B. Exogenous lipoid pneumonia. Clinical and radiological manifestations. Respir Med. 2011;105:659\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorimoto K, Nonaka M, Yamazaki Y, Nakagawa T, Takasaki J, Tsuyuguchi K, et al. Amikacin liposome inhalation suspension for Mycobacterium avium complex pulmonary disease: A subgroup analysis of Japanese patients in the randomized, phase 3, CONVERT study. Respir Investig. 2024;62:284\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKidogawa M, Yamasaki K, Nemoto K, Yatera K. Liposomal Amikacin Inhalation Suspension-induced Pneumonitis. Intern Med. 2022;61:2547\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKohno N. Serum marker KL-6/MUC1 for the diagnosis and management of interstitial pneumonitis. J Med Invest. 1999;46:151\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIshikawa N, Hattori N, Yokoyama A, Kohno N. Utility of KL-6/MUC1 in the clinical management of interstitial lung diseases. Respir Investig. 2012;50:3\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHe J, Zhang J, Ren X. Krebs von den lungen-6 as a clinical marker for hypersensitivity pneumonitis: A meta-analysis and bioinformatics analysis. Front Immunol. 2022;13:1041098.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOhnishi H, Circulating. KL-6 levels in patients with drug-induced interstitial lung disease. Thorax. 2003;58:872\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Amikacin, bronchoalveolar lavage, drug-induced interstitial lung disease, exogenous lipoid, lipoid pneumonia, Mycobacterium avium complex, pneumonia","lastPublishedDoi":"10.21203/rs.3.rs-8234124/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8234124/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAmikacin liposomal inhalation suspension (ALIS), an inhaled aminoglycoside, can effectively treat refractory or relapsed \u003cem\u003eMycobacterium avium\u003c/em\u003e complex lung disease (MAC-LD). However, ALIS is associated with drug-induced interstitial lung disease (DI-ILD), and underlying pathological mechanisms for the same are unclear. This study aimed to determine the aetiology and pathological characteristics of ALIS-associated DI-ILD and assess the impact of ALIS on pulmonary pathology.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThis retrospective cohort study included 25 patients with MAC-LD who received ALIS treatment between September 2021 and October 2024. Clinical data, laboratory findings, high-resolution chest computed tomography results and pathological findings from transbronchial lung biopsy, surgical lung resection and bronchoalveolar lavage were evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOf the 25 patients, 8 developed DI-ILD after a mean duration of 98.5\u0026thinsp;\u0026plusmn;\u0026thinsp;159.8 days following ALIS initiation. Histopathological evaluation showed infiltration of enlarged foamy macrophages containing lipid deposits in patients with and without DI-ILD. In patients with DI-ILD, bronchoalveolar lavage fluid exhibited inflammatory cell infiltration; however, these findings were not diagnostic for a specific entity. Serum Krebs von den Lungen-6 levels significantly increased from baseline to DI-ILD diagnosis (407\u0026thinsp;\u0026plusmn;\u0026thinsp;231 vs. 434\u0026thinsp;\u0026plusmn;\u0026thinsp;250 U/mL; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0422), whereas eosinophil count, C-reactive protein and lactate dehydrogenase levels did not change significantly. Seven of eight patients with DI-ILD exhibited clinical improvement following ALIS discontinuation or dose reduction; one patient required corticosteroid therapy. Of the 8 patients, 5 continued inhalation therapy by switching to every-other-day dosing. The sputum culture conversion rate was maintained even in patients with DI-ILD (63% vs. 47%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.673).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eLipid deposition within the lungs was observed irrespective of DI-ILD, despite no pathognomonic findings being identified for ALIS-associated DI-ILD. Some patients continued therapy by reducing the dosing frequency while preserving therapeutic efficacy even when DI-ILD occurred.\u003c/p\u003e","manuscriptTitle":"Lipid Accumulation and Drug-Induced Interstitial Lung Disease Associated with Amikacin Liposomal Inhalation Suspension in Patients with Mycobacterium avium Complex Lung Disease: A Singlecenter Retrospective Cohort Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 14:22:58","doi":"10.21203/rs.3.rs-8234124/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-16T07:37:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T13:25:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T07:00:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334054234915434908489383371987778384734","date":"2026-01-13T06:24:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205776572129364876454839778404864433738","date":"2026-01-12T07:59:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-03T08:53:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272499287343978924786162059634040725714","date":"2025-12-29T08:20:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-08T17:29:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-03T20:27:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T07:35:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-01T07:34:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pulmonary Medicine","date":"2025-11-29T03:38:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9159522-e8f1-4da3-97dc-9c8690660353","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:05:11+00:00","versionOfRecord":{"articleIdentity":"rs-8234124","link":"https://doi.org/10.1186/s12890-026-04213-2","journal":{"identity":"bmc-pulmonary-medicine","isVorOnly":false,"title":"BMC Pulmonary Medicine"},"publishedOn":"2026-03-09 15:59:47","publishedOnDateReadable":"March 9th, 2026"},"versionCreatedAt":"2025-12-11 14:22:58","video":"","vorDoi":"10.1186/s12890-026-04213-2","vorDoiUrl":"https://doi.org/10.1186/s12890-026-04213-2","workflowStages":[]},"version":"v1","identity":"rs-8234124","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8234124","identity":"rs-8234124","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}