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Value of bronchoalveolar lavage fluid metagenomic next-generation sequencing in acute exacerbation of fibrosing interstitial lung disease: an individualized treatment protocol based on microbiological evidence

BMC Pulmonary Medicine volume 24, Article number: 400 (2024) Cite this article

Abstract

Background

Acute exacerbation of fibrosing interstitial lung diseases (AE-ILD) is a serious life-threatening event per year. Methylprednisolone and/or immunosuppressive agents (ISA) are a mainstay in any regimen, under the premise that pulmonary infection has been promptly identified and controlled. We investigated the value of bronchoalveolar lavage fluid (BALF) metagenomic next-generation sequencing (mNGS) on the treatment adjustment of AE-ILD.

Methods

We conducted a cross-sectional observational study. All data were collected prospectively and retrospectively analyzed. We included fifty-six patients with AE-ILD and nineteen stable ILD who underwent BALF mNGS at the beginning of admission.

Results

Patients with a variety of ILD classification were included. Connective–tissue disease related ILD (CTD-ILD) occupy the most common underlying non-idiopathic pulmonary fibrosis (non-IPF). The infection-triggered AE accounted for 39.29%, with the majority of cases being mixed infections. The microorganisms load in the AE-ILD group was significantly higher. After adjusted by mNGS, the therapy coverage number of pathogens was significantly higher compared to the initial treatment (p < 0.001). After treatment, the GGO score and the consolidation score were significantly lower during follow up in survivors (1.57 ± 0.53 vs. 2.38 ± 0.83 with p < 0.001, 1.11 ± 0.24 vs. 1.49 ± 0.47 with p < 0.001, respectively). Some detected microorganisms, such as Tropheryma whipplei, Mycobacterium, Aspergillus, and mixed infections were difficult to be fully covered by empirical medication. BALF mNGS was also very helpful for excluding infections and early administration of methylprednisolone and/or ISA.

Conclusions

mNGS has been shown to be a useful tool to determine pathogens in patients with AE-ILD, the results should be fully analyzed. The comprehensive treatment protocol based on mNGS has been shown crucial in AE-ILD patients.

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Background

Fibrosing interstitial lung diseases (f-ILDs) are a group of parenchymal pulmonary diseases that are histopathologically characterized by chronic inflammation and fibrosis due to several known and unknown causes. Considering the complex and heterogeneous nature of f-ILDs, their clinical courses are usually unpredictable [1]. Acute exacerbation of f-ILD (AE-ILD) represent an acute event, and often with a poor outcome in various types of f-ILDs, including idiopathic pulmonary fibrosis (IPF) and other types of f-ILDs such as connective–tissue disease related ILD (CTD-ILD) and chronic hypersensitivity pneumonitis (HP) [2, 3]. AE-ILD represents a serious life-threatening event, with a median post-AE survival of only 3–4 months [4]. Acute exacerbations were reduced during the acute phase of the COVID-19 pandemic but now they represent again a major burden [5]. Because of the critical and complex nature of AE-ILD, these critically ill patients always require a multidisciplinary team that involves pulmonologists, rheumatologists, radiologists, laboratory physicians, and pathologists. Clinicians often need to make critical and comprehensive decisions about the treatment of these critically ill patients after considering multiple possible factors.

Given the heterogeneity and complexity of the disease, there are no proven therapies for AE-ILD. In addition to developing effective new drugs, it is critical to accurately estimate the time point of the disease and administer sufficient medication. Corticosteroids are a mainstay in any regimen, and previous historical cohort studies have reported on the positive results of a variety of immunosuppressive agents (ISA), such as cyclophosphamide, cyclosporine A, and tacrolimus, in treating AE-ILD [6, 7]. However, corticosteroids and ISA may inevitably bring some side effects. Pulmonary infection has been shown to be an independent risk factor for death in patients treated with ISA [8]. Empiric antibiotics are also typically recommended until infection can be confidently ruled out, especially for immunocompromised and critically ill patients, for whom early etiological diagnosis is particularly important [9].

The conventional detection methods (CDM) such as traditional microbial culture, polymerase chain reaction (PCR) detection, antigen and/or antibody immunological methods, are time-consuming, have low rates of positive detection, and cannot detect unknown pathogens. With the development of molecular biology, the feasibility of metagenomic next-generation sequencing (mNGS) in etiological detection has recently been demonstrated, especially for the detection of rare, atypical, or slow-growing microorganisms. Our previous studies have proven that the use of mNGS for the detection of lung infections has many advantages over CDM [10,11,12]. However, there are limited literature reports on the clinical application of mNGS in the treatment regimens in AE-ILD.

Previous study has shown C-reactive protein to be a candidate biomarker in f-ILDs, and was associated with poor outcome [13, 14]. Some prospective studies have found that serum procalcitonin can help differentiate between AE-ILD and bacterial infections [15]. The occurrence of a respiratory tract infection serves as an important cause of AE-ILD and promotes disease progression [9]. However, infectious and noninfectious AE-ILD are often clinically indistinguishable. Regarding imaging changes, the newly occurring lesions are superimposed on the basis images of consolidation, exudation, grid, and cable strips that often accompany f-ILDs, which makes it difficult to accurately determine the infectious pathogen by contrast imaging changes alone. Given that it is difficult to distinguish between infectious and noninfectious exacerbations, the item of “exclude acute lung injury caused by infection and other causes” has been discarded from the diagnostic criteria for AE-IPF updated in 2016 [6].

Currently, clinicians often choose empiric anti-infection therapy for AE-ILD, but lack of accurate judgment of microorganisms. This study aimed to assess the value of mNGS of bronchoalveolarlavage fluid (BALF) for the etiological diagnosis of AE-ILD and analyze whether the treatment of AE-ILD could be adjusted based on mNGS results. This study raises the following questions as to whether mNGS may play a role in the analysis of pathogens for AE-ILD; whether there are any pathogens that are difficult to be covered by empirical drugs; whether the specificity of mNGS is affected by changes in the original lung structure of f-ILD and whether treatment strategy could be changed based on mNGS. These questions have not been answered. We hope that this study will help the clinicians accumulate evidence and improve the management of AE-ILD.

Methods

Patients and specimen collection

The present study was a cross-sectional observational study. We prospectively included patients diagnosed with AE-ILD admitted to the Tianjin Medical University General Hospital from September 2019 to June 2023. The patients came from respiratory ward, respiratory intensive care unit and rheumatology intensive care unit. The treatment strategy had undergone multiple consultations with experts from multiple disciplines. The etiology of AE-ILD was classified into infectious and non-infectious, which was comprehensively based on the overall consideration of the clinical characteristics. Infectious etiology was defined as confirmation of bacteria, virus, or fungus in mNGS or CDM detection, which matched the corresponding clinical manifestations (including symptoms), characteristics of respiratory secretions observed under bronchoscopy, HRCT, BALF cellular analysis, blood routine and other laboratory findings, etc. Non-infectious etiology was defined as the lack of clear evidence of microorganisms, and/or the discovery of other factors that may trigger AE. After the patients’ hospitalization, we retrospectively analyzed the causes of AE-ILD during the patient’s follow-up, combining the improvement of clinical symptoms and dynamic changes of HRCT. Patients’ diagnosis was adjusted as necessary. If a patient was repeatedly hospitalized due to acute exacerbation, the first hospitalization data was retained. The major inclusion criteria were as follows: (1) diagnosis of AE-ILD; (2) successfully undergo mNGS testing; (3) patients with complete medical records. The end point of this observational study was 90-day mortality. To clarify the impact of lung structural changes caused by f-ILD on mNGS results, we also included stable ILD patients as the control group, defined as patients with stable imaging and lung function. This study protocol was approved by the Ethical Committee of Tianjin Medical University General Hospital (IRB2020-YX-031-01), and all patients were given informed consent. Our protocol has been registered on ClinicalTrials (NCT 05838183).

All patients received preliminary empirical treatment at admission. Subsequently, patients underwent bronchoscopy within three days of admission. The recovered BALF were separated and sent to mNGS detection, BALF cellular analysis, and the CDM detection. CDM including BALF culture, serum glactomannan test (GM-test), and β-1,3-glucans test (G-test), quantitative real-time polymerase chain reaction (qRT-PCR) for cytomegalovirus (CMV) and Epstein-Barr virus (EBV) detection in blood were performed.

Criteria for the diagnosis of AE-ILD

We defined AE-ILD with the same clinical criteria as AE-IPF, and including both triggered and idiopathic AE-ILDs. The included patients had to meet the following criteria: (1) diagnosis of f-ILD; (2) acute worsening or development of dyspnea < 1 month in duration; (3) high-resolution computed tomography (HRCT) chest imaging demonstrating new bilateral ground-glass opacities and/or consolidation superimposed on a background pattern consistent with fibrotic lung disease; and (4) deterioration not explained by a reversible cause (e.g. fluid overload, thromboembolic disease) [6, 16].All connective tissue disease (CTD)–related ILD patients were referred to rheumatologists before diagnosis. The diagnosis of CTD–ILD was made when the patient had an established autoimmune disease known to cause f-ILD based on previously published criteria [17]. Patients with f-ILD and features suggestive of connective tissue disease CTD, but not meeting the established classification criteria for CTD, were categorized as interstitial pneumonia with autoimmune features (IPAF) [18]. Unclassifiable idiopathic interstitial pneumonia (uIIP), acute interstitial pneumonia (AIP), and idiopathic nonspecific interstitial pneumonia (iNSIP) were diagnosed in accordance with the American Thoracic Society (ATS)/European Respiratory Society (ERS) guidelines [19]. Chronic HP was diagnosed in accordance with the diagnostic criteria by the ATS [20]. A flowchart of participant enrollment criteria was shown in Fig. 1A.

Fig. 1
figure 1

Flow-chart of the study protocol. (A) Flowchart of participant enrollment criteria. (B) Flowchart of study and follow-up protocol. Abbreviations: AE-ILD, acute exacerbation of fibrosing interstitial lung diseases; BALF, broncho-alveolar lavage fluid; HRCT, high-resolution computed tomography; ISA: immunosuppressive agents; mNGS, metagenomic next-generation sequencing

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BALF assessment and specimen sampling

The BALF procedure was done in accordance with the official American Thoracic Society (ATS) clinical practice guideline: the clinical utility of BALF cellular analysis in ILD [21]. The bronchoscope was wedged into the affected lobar or segmental bronchus, targeting the lesion site of new bilateral ground-glass opacities and/or consolidation identified by HRCT. Sterile isotonic saline was instilled in five to ten 20-ml aliquots up to a total volume of 100 to 200 ml. Then, fluid was retrieved using an adjusted negative suction pressure with a target of a minimal volume ≥ 30%. All recovered fluid was aspirated and pooled into a siliconized container. 5 mL of each specimen was separated in a sterile sputum container and kept on ice during transport to the laboratory [22], for mNGS detection, the CDM analyzes and the cytological examination. As for BALF cellular analysis, the cellular components of the recovered BALF were separated through centrifugation at 3000 rpm for 10 min. The total cell count was determined using a hemocytometer, and a differential cell count was obtained on Giemsa-stained cytocentrifuged preparations. Flow cytometric analysis was then performed in a flow cytometer using CD4 and CD8 monoclonal antibodies.

mNGS and analyses

The NGS procedure for the BALF samples included nucleic acid extraction, library construction, sequencing, and information analysis, as previously described [23]. We referred to the previously published standards [12, 24]. For mNGS assay, microorganism detection (bacteria, fungi and viruses) was considered positive if any of the following thresholds were satisfied: (1) the relative abundance of bacteria (excluding Mycobacterium tuberculosis complex) and fungi was greater than 30% at the genera level; (2) a pathogen considered to be positively detected using a traditional detection method had the mNGS reads number greater than 50; (3) for Mycobacterium tuberculosis complex, at least one read had to be aligned to the reference genome at the species or the genera level; and (4) virus detection was considered if the read number was 3 or greater.

Propionibacterium acnes, Micrococcus luteus, Malassezia globosa, Lactococcus lactis, Saccharomyces, Torque teno virus, parvovirus, Ureaplasma, Staphylococcus epidermidis, intestinal colonized flora and anaerobic bacteria were considered colonizing microorganisms [25]. These microorganisms could normally colonize the in respiratory tract, and may also serve as pathogens that induce AE-ILD. If the characteristics of microorganisms detected by mNGS were consistent with patients’ clinical symptoms, underlying diseases, imaging characteristics, and laboratory indicators, they were defined as pathogenic microorganisms. On the contrary, they were defined as colonized microorganisms or false positives results. Pathogenic microorganisms were ascertained by two specialist clinicians based on the comprehensive assessments which consisted of the number of reads for mNGS, the clinical presentation, radiological manifestations, conventional detection findings, and the clinical epidemiology. A third senior clinician and a fourth clinical microbiologist were further involved in the discussion in case of a major disagreement between the first two clinicians. Finally, treatment adjustments were made based on the comprehensive analysis of the mNGS results.

Follow up and HRCT scoring

A flowchart of study and follow-up protocol was shown in Fig. 1B. Survivors underwent HRCT scans at the onset and within 90 days after treatment. No AE occurred within the interval between two HRCT scans. The researchers scored the HRCT images by evaluating three images taken at the levels of the aortic arch, carina, and 1 cm above the diaphragm according to the previously described protocol [26]. Each lobe of the lung was scored at a scale of 0–5 for both alveolar and interstitial abnormalities, depending on the degree of involvement of each lobe and the type of images. Ground-glass opacity represented the alveolar findings (GGO score), while reticular and cellular opacities represent interstitial presentation (Fibrosis score). In addition, as consolidation is also a common finding in CTD-ILD, we have added the Consolidation score. The scoring criteria were as the following: one point for GGO/ fibrosis/consolidation 5%, two points for 6–25%, three points for 26–50%, four points for 51–75%, and five points for 76%. Take all scores in each lobe of both lungs and take the average. Average the scores of each lung lobe among all three experienced respiratory physicians for data analysis.

Statistical analysis

Count data were expressed as the percentage of the number of cases (n%). Results for variables were expressed as the means ± standard deviations (SD). Comparisons of the two rates were performed using the Chi-square test. Comparisons of two independent variables were made using Student’s t test. Differences were statistically evaluated by a one-way ANOVA followed by Fisher’s PLSD. Error bars were used to indicate the SD. All statistical analyses were done used SPSS 28.0 (IBM, New York, NY, USA). Differences were considered statistically significant at p values of < 0.05.

Result

Baseline characteristics

A total of 75 patients who underwent BALF mNGS at the beginning of admission (D1-3), including 56 patients with AE-ILD and 19 stable ILD. The etiology of patients with AE-ILD were IPF (n = 8), chronic HP (n = 4), IPAF (n = 10), AIP (n = 1), iNSIP (n = 6), uIIP (n = 1), and CTD-ILD (n = 26). The most common non-IPF was CTD-ILD (46.43%). Regarding the classification of CTD, there were 9 cases of antisynthetase syndrome (ASS), 7 cases of rheumatoid arthritis (RA), 6 cases of dermatomyositis (DM), 1 cases of polymyositis (PM), 1 case of anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), 2 case of and mixed connective tissue disease (MCTD). Among CTD-ILD, 10 cases were diagnosed before hospitalization, and 16 cases (61.54%) were newly diagnosed after admission because of acute exacerbation. The usual interstitial pneumonia (UIP) pattern of HRCT occupied 58.93%(33/56) in all AE-ILD. The 90-day mortality was 26.79% (15/56). The infection-triggered AE accounted for 39.29%(22/56). The clinical characteristics of the included patients were summarized in Table 1.

Table 1 Baseline characteristics of AE-ILD
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Spectrum of microorganism detected by mNGS

We compared the species of microorganism in the AE-ILD and stable ILD detected by mNGS. The compositions of microorganisms detected by mNGS were shown in Fig. 2. mNGS showed a higher microorganisms load in the AE-ILD group than stable ILD. Species of microorganisms were significantly higher in the AE-ILD compared with stable ILD (1.59 ± 1.85 vs. 0.37 ± 0.60, p < 0.001) (Fig. 3A); Species of microorganisms were significantly higher in patients with the UIP pattern compared with non-UIP (2.09 ± 2.02 vs. 0.87 ± 1.29, p = 0.009) (Fig. 3B); Species of microorganisms were significantly higher in the immunosuppressed patients compared with non-immunosuppressed. (2.43 ± 2.17 vs. 1.31 ± 1.66, p = 0.049) (Fig. 3C); No statistical difference was found between the non-survivors compared with survivors. (2.31 ± 2.06 vs. 1.37 ± 1.75, p = 0.11) (Fig. 3D). Further details were shown in Supplementary Table S1.

Fig. 2
figure 2

Spectrum of microorganisms identified by mNGS. X-axis represented number of cases

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Fig. 3
figure 3

Distribution of microorganism species in different groups. (A) Species of microorganisms were significantly higher in the AE-ILD compared with stable ILD. (1.59 ± 1.85 vs. 0.37 ± 0.60, p < 0.001); (B) Species of microorganisms were significantly higher in patients with the UIP pattern compared with non-UIP. (2.09 ± 2.02 vs. 0.87 ± 1.29, p = 0.009); (C) Species of microorganisms were significantly higher in the immunosuppressed patients compared with non-immunosuppressed. (2.43 ± 2.17 vs. 1.31 ± 1.66, p = 0.049); (D) No statistical difference was found between the non-survivors compared with survivors. (2.31 ± 2.06 vs. 1.37 ± 1.75, p = 0.11). * p < 0.05, ** p < 0.01, *** p < 0.001

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Distribution and the coverage rates of microorganisms in infectious AE-ILD

Among all AE-ILD patients, infection was considered as a contributing factor to trigger AE in 22 (39.29%) cases. After removing colonizing organisms, 8 cases were (36.36%) tested positive for a single microorganism and 14 cases (63.64%) for two or more microorganisms (Fig. 4A). The detected microorganisms were: (1) viruses (16 cases), including Human-betaherpesvirus-5, Human-gammaherpesvirus-4, Human-alphaherpesvirus-1, Human-betaherpesvirus-7, Human_orthopneumovirus, Human_polyomavirus_4, Rhinovirus_B, Rhinovirus_A, Influenza_B_virus, Severe_acute_respiratory_syndrome-related_coronavirus; (2) bacteria (12 cases), including Acinetobacter_baumannii, Pseudomonas aeruginosa, Enterobacter_hormaechei, Klebsiella_aerogenes, Klebsiella_pneumoniae, Nocardia_abscessus, Tropheryma_whipplei, Methylobacterium_radiotolerans, Mycobacterium_avium_complex_(MAC), Mycobacterium_tuberculosis_complex; and (3) fungi (11 cases), including Pneumocystis jirovecii, Aspergillus_flavus, Aspergillus_fumigatus, Candida_albicans, Candida_parapsilosis, Candida glabrata, Pichia_kudriavzevii. The mixed microorganism distribution in infectious AE-ILD was shown in a Wayne diagram (Fig. 4B). The intersection of the Wayne diagram indicated the number of cases with two or three microorganisms present simultaneously. Detection of microorganism in infectious AE-ILD was shown in Fig. 5 (mNGS vs. CDM).

Fig. 4
figure 4

Etiological distribution of AE-ILD. (A) Pie chart showed the etiological of AE-ILD. (B) Wayne diagram showed the distribution of viruses, bacteria, and fungi in infectious AE-ILD cases

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Fig. 5
figure 5

Detection of microorganism in infectious AE-ILD. (mNGS vs. CDM). CDM, conventional detection methods; mNGS: metagenomic next-generation sequencing

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In the cases of infectious AE-ILD, the initial and treatment adjusted antimicrobial coverage species for microorganisms were 9 (15%), 34 (59%), respectively. 15 (26%) species were not covered (Fig. 6A). The initial empirical antimicrobial coverage rates for viruses, bacteria, and fungi were 12.9%, 25%, and 20%, respectively. After adjusting the treatment based on mNGS findings, the overall coverage rates were 58.06%, 100%, 100%, respectively. All were significantly higher compared to the initial coverage rates (p < 0.001) (Fig. 6B). The detailed species of total detected pathogens, species covered by initial treatment, and species covered by adjusted treatment were shown in Fig. 6C. The following microorganisms were not therapeutic covered: Human-betaherpesvirus-5 (n = 3), Human-gammaherpesvirus-4 (n = 5), Human-alphaherpesvirus-1 (n = 2), Human_ betaherpesvirus_7 (n = 1), Human_orthopneumovirus (n = 1), Human_ polyomavirus_4 (n = 1), Rhinovirus A (n = 1). The reason may be that the current treatment of viral infections is largely supportive.

Fig. 6
figure 6

The coverage of antimicrobial therapy for microorganisms in infectious AE-ILD. (A) The initial and treatment adjusted antimicrobial coverage species for pathogens were 9 (15.79%), 35 (61.40%), respectively. 13 (22.81%) species were not covered. (B) After adjusting the treatment based on mNGS findings, the overall therapeutic coverage rates of viruses, bacteria, and fungi were 58.06%, 100%, 100%, respectively. All were significantly higher compared to the initial coverage rates (p < 0.001) *** p < 0.001. Y-axis represented species of pathogens. (C) The detailed species of total detected pathogens, species covered by initial treatment, and species covered by adjusted treatment

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Antimicrobial treatment adjustments based on mNGS findings

The bacteria included: Tropheryma whipplei (no. 11), Acinetobacter baumannii (nos. 3 and 18), Klebsiella aerogenes (no. 7), Klebsiella pneumoniae (nos. 10, 13, 18, and 21), Pseudomonas aeruginosa (no. 9, and 20), Nocardia_abscessus (no. 10), Methylobacterium_radiotolerans (nos. 17, 18), Enterobacter_hormaechei (no. 21), Mycobacterium_avium_complex_(MAC) (no. 17), and Mycobacterium_tuberculosis_complex (no. 21). The treatment was adjusted based on the finding of the following fungi: Pneumocystis jirovecii (nos. 1, 2, 12, and 15), Candida albicans (no. 6), Candida parapsilosis (no. 18), Pichia kudriavzevii (no. 7), Aspergillus flavus (no. 8), and Aspergillus fumigatus (nos. 14 and 17). In 9 cases (nos. 4, 6, 7, 8, 9, 10, 12, 16, and 17), treatment was adjusted based on the finding of virus. Cases of adjustment of antimicrobial treatment are shown in Table 2.

Table 2 Antimicrobial treatment adjustments based on mNGS findings
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Despite of the initial empiric antimicrobials treatment, some microorganisms that have not been covered by conventional antimicrobials therapy need to be given special attention by clinicians. Among the enrolled patients in this study, Tropheryma whipplei was not uncommon. In case (no. 11), Tropheryma whipplei was considered triggering AE and lower respiratory tract infection. Ceftriaxone was added for targeted treatment of Tropheryma whipplei and the patient’s symptoms were significantly improved. There was no intervention for Tropheryma whipplei in case (no. 45) due to the improved symptoms under the original treatment and the stable HRCT imaging during follow-up. In stable ILD, Tropheryma whipplei was also detected (no. 58) (Supplementary Table S1). Mycobacterium, Aspergillus, and mixed infections were usually secondary occurred in those who with immunosuppressed, and with severe lung structure damaged. The imaging changes of treatment of mixed infections (no. 17) were shown in Fig. 7A.

Fig. 7
figure 7

Comparison of HRCT on-admission and after treatment. (A) The comparison of HRCT before and after treatment of mixed infections (no. 20). CT images showed significant improvement of infiltration after treatment (right) compared with that before treatment (left). (B) Comparison of CT score on-admission and after treatment in survivors. The GGO score was significantly lower after treatment (1.57 ± 0.53 vs. 2.38 ± 0.83, p < 0.001); the consolidation score was significantly lower (1.11 ± 0.24 vs. 1.49 ± 0.47, p < 0.001); the fibrosis score showed no statistical differences (2.07 ± 0.97 vs. 2.25 ± 0.83, p = 0.357), indicating an improvement in clinical condition

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Negative mNGS findings as an auxiliary for excluding infection

Non-infectious AE-ILD accounted for 34 cases, among which mNGS was positive in 13 cases. These microorganisms included Neisseria mucosa (n = 1), Rothia_ mucilaginosa (n = 1), Prevotella melaninogenica (n = 2), Prevotella jejuni (n = 1), Prevotella intermedia (n = 1), Streptococcus pneumonia (n = 1), Streptococcus mitis (n = 1), Staphylococcus_epidermidis (n = 1), Lautropia_mirabilis (n = 1), Corynebacterium propinquum (n = 1), Tropheryma whipplei (n = 1), Human-gammaherpesvirus-4 (n = 1), Human-betaherpesvirus-7 (n = 2), Candida glabrata (n = 1), Candida_albicans (n = 1), Aspergillus_flavus (n = 1). Most of them were colonizing microorganisms. These colonizing or false positive microorganisms were excluded based on the judgment criteria described above.

In 12 cases, immunomodulatory were used after infection was ruled out by mNGS (Table 3). There were 7 cases of CTD-ILD, 4 cases of IPAF, 1 cases of iNSIP. Importantly, most patients had no history of CTD-ILD or IPAF before admission. 5 cases (nos. 29, 31, 43, 46, and 47) added methylprednisolone, 7 cases (nos. 23, 24, 27, 28, 36, 40, and 48) increased the dosage of methylprednisolone. 5 cases (nos. 28, 36, 40, 46, and 47) added ISA. There was no decrease in the use of antimicrobials, mainly due to the complexity of these critically ill patients and vigilance for secondary infections following methylprednisolone.

Table 3 Methylprednisolone/ISA adjustment based on negative mNGS findings
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Comprehensive treatment protocol based on mNGS

For critically ill patients with uncontrolled autoimmune factors and infection, immunoregulation and anti-infection are equally important parts of treatment. Based on the pathogens finding of mNGS, the antimicrobial drugs were adjusted accompanied by increasing of methylprednisolone (nos. 11, 13 and 15), which was due to the unstable rheumatic immune indicators. For such patients, early and appropriate use of methylprednisolone may be the most beneficial, but the premise is that the infection must be fully controlled. In one patient (no. 21), in addition to adjust effective antimicrobial therapy based on mNGS, methylprednisolone was appropriately reduced while RA was stable, owing to the protocol of focusing on infection control. mNGS has played an important role in adjusting the direction of comprehensive treatment for these patients.

Treatment effectiveness and prognosis

The 90-day mortality was 26.79% (15/56). All survivors underwent HRCT scans within 90 days after treatment and compared to the image at the onset. No AE occurred within the interval between two HRCT scans. All survivors showed different degrees of clinical symptoms improvement (fever, dyspnea, and cough). We compared changes of CT score on-admission and after treatment in survivors. After treatment, the GGO score was significantly lower during follow up (1.57 ± 0.53 vs. 2.38 ± 0.83, p < 0.001); the consolidation score was significantly lower (1.11 ± 0.24 vs. 1.49 ± 0.47, p < 0.001); the fibrosis score showed no statistical differences (2.07 ± 0.97 vs. 2.25 ± 0.83, p = 0.357), indicating an improvement in clinical condition (Fig. 7B).

Discussion

Despite many studies on AE-ILD, most of the treatment options are based on clinical experience. Few studies have targeted treatment approach based on accurate etiological analysis [27, 28]. Our study discussed the important clinical significance of mNGS for the treatment adjustment in patients with AE-ILD and provided more evidence for the empirical treatment of AE-ILD.

Many studies have confirmed that increased microbial load can accelerate the progression of pulmonary interstitial fibrosis [29], which is consistent with our findings. There are also a growing number of in vivo experiments elucidating the mechanism [29, 30]. More common respiratory viral pathogens, including respiratory syncytial virus and cytomegalovirus, have also been detected during exacerbations using molecular techniques [31]. Theoretically, mNGS can detect all pathogens with known genome sequences. Currently, there are more than 8,000 known pathogens that can be detected with high sensitivity [12]. In the present research, although most microorganisms could have covered by empiric antimicrobials, some rare microorganisms have been reported. One study has also reported Tropheryma whipplei detected through nanopore sequencing in three patients with ILD [32]. Research on the triggering of AE-ILD by rare microorganisms needs to be further carried out; after all, these microorganisms cannot be detected through conventional examinations. Moreover, anti-Aspergillus and anti-tuberculosis drugs are not routine initial treatment, especially when AE was as a major imaging manifestation. Some of the detected microorganisms were regarded as colonizing organisms. However, some colonizing microorganisms, such as Haemophilus, Neisseria, and Streptococcus species, have been shown to play an important role in AE-ILD microbiota disturbance and have been validated in animal experiments [29, 30, 33, 34]. The mechanism of normal skin microbiota and microbial translocation and dysbiosis in the oral or respiratory tract has been shown to induce AE-ILD [29]. Therefore, the clinical value of colonizing organisms should also be given sufficient attention. With the advantages of mNGS for unknown disease detection, we have shown that the spectrum of respiratory pathogens may induce AE-ILD, which is much more valuable than blood samples [35]. Although a large number of patients in this study had already been treated with antibiotic regimens at the time of onset, initial empirical antimicrobial treatment could not cover all microorganisms promptly without the mNGS findings. Our research indicated the complexity of microorganism infections in AE patients. Whether these microorganisms are the initiating factors, or secondary to the patient’s consumption during AE, is one of the key issues that future clinical and basic research needs to focus on exploring.

It is noteworthy that CTD-ILD and IPAF formed the largest groups in our study. Consistent with our research results, previous study has shown that many patients do not have a clear history prior to admission to the intensive care unit for exacerbations, and CTD is frequently diagnosed during the intensive care unit stay [36] AE-ILD can be the first manifestation of f-ILD in these patients [37]. Timely and appropriately immunomodulation is particularly important for AE-ILD, especially for patients with a first diagnosis of CTD after admission. mNGS has great value for ruling out infection in these patients. A randomized, double-blind, placebo-controlled phase 3 trial showed that adding intravenous cyclophosphamide pulses to glucocorticoids increased the 3-month mortality rate in patients with acute exacerbation of IPF. Infection was one of the main adverse events [38]. This indicates that the interaction between microorganisms and hosts is not only crucial in the occurrence of acute exacerbation, but also a key factor in the treatment process of AE-ILD. Obviously, “A one-size-fits-all approach” to the management of this group of diseases is not appropriate. Accurately grasping the relationship between microorganisms and host immunity and timely adjusting therapeutic plan may play a key role during treatment.

The importance of BALF in AE-ILD has been a controversial topic in recent years, although BALF has been considered necessary to exclude an infectious aetiology [39]. For the diagnosis of AE-ILD, due to the challenge in distinguishing between infection and non-infection, the current guidelines for AE-IPF diagnosis no longer require exclusion of infection [6]. Referring to the criteria of AE-IPF, it is permissible to diagnose AE-ILD without undergoing bronchoscopy and BALF. For the treatment of AE-ILD, broad-spectrum antibiotics are often given during the initial treatment. At present, most studies on the classification BALF cells have only confirmed their prognostic significance [27, 40]. In the previous viewpoints, the controversial reason for performing bronchoscopy on AE-ILD patients is that it may not change the treatment strategies, and thus the necessity has been questioned [39]. The important finding of our study indicated the results of BALF mNGS in patients with AE-ILD provided new clues for treatment, which have not been elucidated in previous studies. The necessity of BALF for AE-ILD needs to be reassessed.

Bronchoscopy is the most commonly used technique in the field of respiratory disease intervention [41, 42]. The incidence of adverse events typically depends on the skills, abilities, and experience of bronchoscopy procedures, which may hinder the widespread applicability in critically ill patients. Nonetheless, many studies have shown the benefits of bronchoscopy in critically ill patients. Bulpa, P. A et al. have demonstrated that the combination of BAL and transbronchial lung biopsy (TBLB) has excellent diagnostic and therapeutic value for patients with unexplained pulmonary infiltration in the ICU undergoing mechanical ventilation, and there is generally no bronchoscopy related mortality [43]. Our previous published studies have also shown safe and accurate diagnosis and treatment of critically ill patients through bronchoscopy [44, 45]. BAL has been considered a relatively safe and well-tolerated procedure during AE-ILD in some studies [27, 40, 46, 47], however, it is undeniable that the safety of bronchoscopy operation during AE-ILD is also an issue that needs to be fully considered. Although there were no complications associated with bronchoscopic procedures in our study, not all AEs are suitable for bronchoscopy. A comprehensive weighing of the pros and cons was necessary.

Due to the complexity of the causes of AE-ILD, there is still a long way to go to overcome this disease. In addition to the usage of two anti-fibrotic drugs that have been proven to slow disease progression, immunomodulatory therapy plays a crucial role in helping patients overcome the acute phase [6, 7]. More and more recent studies have shown the importance of immune regulation in IPF, which also provides clues for targeted treatment of f-ILD patients [48]. A recent study also characterized the transcriptome landscape and morphological progression of fibrosis of fHP. The interaction between immune cell types and resident cells in the lung microenvironment may ultimately determine the degree and likelihood of fibrosis resolution [49]. This will become a new anti-inflammatory and immunotherapy method for AE-ILD. As with the advancement of detection technology, the current results have brought unprecedented information and personalized treatment approach of AE-ILD. A large amount of research data has laid the foundation for establishing an individualized treatment, including the inter-individual molecular, genetic and genomic differences, a large number of diagnostic, prognostic and theragnostic biomarker candidates in f-ILD [50]. Although the transition from these basic research into clinical practice remains to be a challenge, performing bronchoalveolar lavage fluid analysis on AE-ILD patients will provide further information beyond the microorganism, which could be applied to personalized treatment.

This article provided microorganisms evidence and introduced a rapid individualized treatment approach based on mNGS results for AE-ILD patients. Further research in identifying the etiology is vital to balance the ISA and antibiotic treatment for these critically ill patients. It is hoped that the development of molecular biology technology and the accumulation of evidence from clinical research will offer the potential for improved drug selection and development to realize patient-centric precision medicine for AE-ILD.

Conclusions

mNGS detected some microorganisms that were difficult to cover with empirical medication, which needs to be taken seriously by clinicians. mNGS was also very helpful for excluding infections and early administration of methylprednisolone and/or ISA. The mNGS-based treatment adjustment has been shown crucial in AE-ILD patients, the results should be fully analyzed in conjunction with clinical practice.

Data availability

The data that support the findings of this study have been deposited into NCBI, with accession numbers PRJNA1058145, PRJNA1051816, PRJNA1048156.

Abbreviations

AE-ILD:

Acute exacerbation of fibrosing interstitial lung diseases

AIP:

Acute interstitial pneumonia

ANCA-AAV:

Anti-neutrophil cytoplasmic antibodies-associated vasculitis

ASS:

Antisynthetase syndrome

ATS:

American Thoracic Society

BALF:

Broncho-alveolar lavage fluid

CDM:

Conventional detection methods

CMV:

Cytomegalovirus

CTD-ILD:

Connective–tissue disease related interstitial lung diseases

DM:

Dermatomyositis

EBV:

Epstein-Barr virus

ERS:

European Respiratory Society

GGO:

Ground-glass opacity

GM-test:

Glactomannan test

G-test:

β-1,3-Glucans test

HP:

Hypersensitivity pneumonitis

HRCT:

High-resolution computed tomography

iNSIP:

Idiopathic nonspecific interstitial pneumonia

IPAF:

Interstitial pneumonia with autoimmune features

IPF:

Idiopathic pulmonary fibrosis

ISA:

Immunosuppressive agents

MCTD:

Mixed connective tissue disease

mNGS:

Metagenomic next-generation sequencing

PM:

Polymyositis

qRT-PCR:

Quantitative real-time polymerase chain reaction

RA:

Rheumatoid arthritis

uIIP:

Unclassifiable idiopathic interstitial pneumonia

UIP:

Usual interstitial pneumonia

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Acknowledgements

We thank all patients for cooperating with the follow-up, and all medical staff involved in the treatment and care of patients.

Funding

National Natural Science Foundation of China(82300120, 82170097, 81970083, 81570084, 81270144, 30800507). The Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-008A).

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  1. Siyu Zhan and Shuo Li contributed equally to this work.

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  1. Department of Respiratory and Critical Care Medicine, Tianjin Medical University General Hospital, Tianjin, 300052, China

    Siyu Zhan, Shuo Li, Yaoqian Cao, Dan Liu & Jing Feng

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Contributions

FJ and LD contributed to study conception and design. LS, CYQ and ZSY drafted and revised the manuscript. ZSY was responsible for collecting clinical data from medical records. LD was responsible for following up with patients and analyzing the effects of treatment. LD performed the statistical analyses. All authors read and approved the final manuscript.

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Correspondence to Dan Liu or Jing Feng.

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This study was carried out in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Review Committee of Tianjin Medical University General Hospital. All of the patients included provided written informed consent.

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Zhan, S., Li, S., Cao, Y. et al. Value of bronchoalveolar lavage fluid metagenomic next-generation sequencing in acute exacerbation of fibrosing interstitial lung disease: an individualized treatment protocol based on microbiological evidence. BMC Pulm Med 24, 400 (2024). https://doi.org/10.1186/s12890-024-03216-1

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BMC Pulmonary Medicine

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