Advantages of metagenomic next-generation sequencing in the management of ANCA-associated vasculitis patients with pulmonary infections as a rule-out tool

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It is sometimes difficult to differentiate pulmonary infection from pulmonary involvement of vasculitis in AAV patients. Fiberoptic bronchoscopy and bronchoalveolar lavage fluid (BALF) assays are useful diagnostic methods. In addition to conventional microbiological tests (CMTs), metagenomic next-generation sequencing (mNGS) facilitates rapid and sensitive detection of various pathogens. The current study aimed to evaluate the advantages of additional BALF mNGS in the management of pulmonary infection in AAV patients. Methods: 27 patients with active AAV and suspected pulmonaryinfection whose BALF samples were tested by mNGS and CMTs and 17 active AAV patients whose BALF were tested by CMTs alone were retrospectively recruited. The results of microbiological tests and adjustments of treatment upon BALF mNGS were described. The durations of antimicrobial treatment and in-hospital mortality in patients were compared. Results: Among the 27 patients whose BALF samples were tested by mNGS, 25.9% of patients did not have any evidence of pathogenic microorganism in their BALF samples, and 55.6% of patients had polymicrobial infections, including bacteria, fungi and virus, while 40.7% of patients did not have any evidence of pathogenic microorganism in their BALF and serum samples according to CMTs tests. Patients in the BALF mNGS group received significantly shorter duration of antibacterial and total antimicrobial treatment than patients in the CMT alone group (17.3±14.7 vs. 27.9±19.0 days, P =0.044; 18.9±15.0 vs. 29.5±17.7 days, P =0.040, respectively). Patients in the BALF mNGS group had significantly lower in-hospital mortality than patients in the CMT alone group (4/27 vs. 7/17, P =0.049). Conclusion: Compared with CMT alone, additional mNGS tests may shorten the duration of antimicrobial treatment and decrease death from severe infection by providing precise and quick diagnosis of infection. antineutrophil cytoplasmic antibody vasculitis pulmonary involvement pulmonary infection metagenomic next-generation sequencing Figures Figure 1 INTRODUCTION Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) comprises granulomatosis with polyangiitis (GPA, previously known as Wegener’s granulomatosis), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA, previously known as Churg-Strauss syndrome) ( 1 , 2 ). The lung and kidney are the two most involved organs in AAV. Pulmonary involvement occurs in 25–80% of AAV patients ( 3 – 6 ), including interstitial lung disease, cavity, nodules, and alveolar hemorrhage, and diffuse alveolar hemorrhage (DAH) is one of the most severe manifestations of AAV ( 7 ), significantly contributing to morbidity and mortality. AAV is usually fatal when left untreated, with a 1-year mortality rate of approximately 80% ( 8 ). Although the introduction of immunosuppressive therapy substantially improved the outcome of AAV, it was also associated with high rates of adverse events, especially secondary infection ( 9 ). Consistent with studies from other groups ( 10 , 11 ), we found that secondary infection was the leading cause of death during the first year after the diagnosis of AAV ( 12 ), and the majority of infectious complication was pulmonary infection ( 13 ). Therefore, early recognition and appropriate treatment in time are of great importance. However, it is sometimes difficult to differentiate pulmonary infection from pulmonary involvement of vasculitis in AAV patients due to similar clinical manifestations (eg. fever, cough and hemoptysis), laboratory findings (eg. elevated white blood cell and neutrophil counts in peripheral blood, C-reactive protein [CRP] and erythrocyte sedimentation rate [ESR]) and imaging features (patches, masses, ground-glass opacities and consolidations). Moreover, given that the treatment strategies for pulmonary involvement of active AAV and pulmonary infection are inconsistent, the hard differential diagnosis presents us with difficulties in treatment decisions. Misdiagnosing pulmonary infection as active AAV pulmonary involvement and thus treating with aggressive immunosuppressive therapy could probably result in disastrous consequences of aggravation of infection. Fiberoptic bronchoscopy and bronchoalveolar lavage fluid (BALF) assays are useful diagnostic methods when a co-existing infection is suspected in AAV patients with pulmonary involvement. In addition to conventional microbiological tests (CMTs) of BALF, metagenomic next-generation sequencing (mNGS) is a nucleic acid sequencing technique with a high-throughput capacity and an approach that can theoretically detect any microorganism in the bio-sample, including BALF ( 14 , 15 ). Various studies have reported the application of mNGS to detect pathogens in pulmonary infection ( 16 – 19 ). In the study by Peng et al., which involved immunocompromised patients with pulmonary infection, it was found that the diagnostic performance of BALF mNGS was similar to that of comprehensive CMTs, but when there is a lack of consideration of potential pathogens, the combination of mNGS and CMTs might be a better diagnostic strategy ( 18 ). To our knowledge, there is few, if any, reports on the application of mNGS in AAV. Thus the current study evaluated the advantages of additional BALF mNGS in the management of pulmonary infection in patients with AAV. MATERIALS AND METHODS Patients AAV patients diagnosed at Peking University First Hospital between March 2019 and March 2022 were reviewed. Treatment protocols were described previously ( 20 ). In brief, patients received the induction therapy typically including corticosteroids in combination with cyclophosphamide (CTX) or rituximab (RTX). Patients with severe pulmonary hemorrhage or acute renal failure requiring dialysis at diagnosis received additional methylprednisolone pulse and plasmapheresis. For maintenance therapy, daily oral azathioprine (AZA) or RTX was administered. Those who were clinically suspected of pulmonary infection and underwent fiberoptic bronchoscopy were retrospectively recruited. All patients met the Chapel Hill Consensus Conference criteria for AAV ( 1 ). Patients with secondary vasculitis or with other comorbid renal diseases were excluded. 27 patients whose BALF samples were simultaneously available for mNGS tests besides CMTs (termed the BALF mNGS group), and 17 patients whose BALF was only tested by CMTs (termed the CMT alone group), were included in the study. This research was conducted in accordance with the Declaration of Helsinki and was approved by the Clinical Research Ethics Committee of the Peking University First Hospital. Written informed consent was obtained from the patients or their guardians. Data collection All the clinical and laboratory data were collected from the medical records of the patients at admission and at the time of bronchoscopies, including age, sex, vital signs, diagnosis, ANCA serotype, disease phenotype, organ involvement, laboratory data (serum creatinine, CRP, ESR, white blood cell [WBC], neutrophil and lymphocyte counts, hemoglobin, platelet count, CD4 + T cells, serum albumin, lactate dehydrogenase [LDH] and arterial blood gas analysis), immunosuppressive therapies, antimicrobial treatment, concomitant treatment, respiratory support, response to treatment and intensive care unit (ICU) stay. The estimated glomerular filtration rate (eGFR) was calculated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) Eq. (21). The risk scores CRB65 were calculated based on the initial available data, which involve mental status, respiratory rate, blood pressure and age ( 22 ). Specimen collection All bronchoscopies were performed by experienced bronchoscopists. The target lesion was decided according to the chest CT beforehand. A standard bronchoalveolar lavage was collected before any other bronchoscopy procedures to avoid possible contamination. Once collected, BALF specimens were immediately sent to the clinical microbiology laboratory for CMTs and to a commercial laboratory for mNGS. Conventional microbiological tests Comprehensive CMTs were performed on BALF, serum, sputum and throat swab samples, including culture, antigen detection, multiplex PCR, and direct microscopic examination of specimens according to standard procedures ( 23 , 24 ). Gram staining, acid-fast staining, hexamine silver staining, the galactomannan antigen detection test (GM test), cryptococcal capsular polysaccharide antigen test and Aspergillus antibody detection with BALF samples, interferon-gamma release assay (T-SPOT. TB), ( 1 , 3 )-β-D-glucan test (G test) and GM test with serum samples were performed to identify bacteria, including Mycobacterium tuberculosis , and fungi, including Pneumocystis carinii , Cryptococcus neoformans, Candida and Aspergillus . mNGS of BALF mNGS was performed once the sample was obtained. The total turnaround time is approximately 24 hours (5 hours for shipping and 19 hours for testing). BALF samples were collected from patients for metagenomic analysis pipeline for microbial identification (MAPMI). Total DNA and RNA were extracted from clinical samples by using a MAPMI sample preparation kit (CapitalBio Technology, Beijing, China. Catalogue No. S60150) according to the manufacturer’s instructions. The extracted RNA was reverse-transcribed using random primers, and cDNA was pooled with DNA from the same clinical sample for sequencing library preparation. The pooled nucleic acid was enzymatically fragmented to a size of 200–300 base pairs (bp), and sequencing libraries were constructed through end repair, adapter ligation and PCR amplification. Sequencing templates were further prepared with the OneTouch2 System (Life Technologies, CA, USA) and sequenced on the BioelectronSeq 4000 sequencer (CapitalBio Technology) after quality control. A negative control sample of water was used in each run to monitor potential contamination. The original sequencing data were subjected to quality control, and reads with lengths less than 50bp, low quality, or low complexity were removed. The remaining high-quality sequencing data were mapped to the human reference genome grch38 for the depletion of human host sequences using Bowtie2 software. Subsequently, the nonhuman sequences were classified by simultaneous alignment to the genomic sequence databases downloaded from the NCBI and PATRIC databases. To judge the suspected pathogens in clinical samples, we reviewed data from different types of samples from healthy people and calculated the relevant reference values, including the hit read number and coverage of all bacteria, fungi, viruses and parasites detected. Moreover, pathogens detected in the negative control sample of water were removed from the results of clinical samples. The final pathogen detection results included a list of suspected pathogens, the number of hit reads (using the term reads per ten million [RPTM]) and genome-level coverage statistics. Statistical analysis Data were expressed as the mean ± SD (for data that were normally distributed) or median and interquartile range (IQR; for data that were in skewed distribution) for continuous variables and number (%) for categorical variables as appropriate. Quantitative parameters were compared using Student’s t test (for data that were normally distributed) or the nonparametric test (for data that were in skewed distribution) as appropriate. Categorical variables were compared using the χ 2 test. A P value < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS 18.0 (Chicago, IL, USA). RESULTS General data of the AAV patients The clinical and laboratory data of the patients in the BALF mNGS group and CMT alone group are presented in Table 1 . No significant difference in age, sex, laboratory data, the ratio of partial pressure to fraction of inspiration oxygen (PaO2/FiO2), CRB-65, or immunosuppressive regimens was found between these two groups of patients. Therefore, there was no significant difference in disease activity of AAV, severity of infection or intensity of immunosuppressive therapy between the two groups. Table 1 General data of the patients in the BALF mNGS group and CMT alone group Items BALF mNGS group (n = 27) CMTs group (n = 17) P values Age, years 66.1 ± 9.2 61.6 ± 18.5 0.371 Male, n (%) 11(41%) 11 (65%) 0.126 MPO-ANCA/PR3-ANCA, n 18/9 12/5 0.786 ANCA titers, AU/ml 122.8 ± 111.1 108.3 ± 87.8 0.657 Serum creatinine, umol/l 329.4 ± 232.7 342.7 ± 249.0 0.858 eGFR, ml/min/1.73m 2 30.2 ± 31.2 30.3 ± 28.7 0.995 CRP, mg/l 31.6 (13.5/90.5) 41.0 (5.0/107.2) 0.990 ESR, mm/1h 69.1 ± 44.8 58.3 ± 33.5 0.642 PCT, ng/ml 0.34 (0.13/1.52) 0.37 (0.14/1.34) 0.951 WBC, *10 9 /l 11.4 ± 6.1 9.7 ± 3.8 0.292 Neutrophils, *10 9 /l 9.8 ± 6.0 8.0 ± 3.6 0.256 Lymphocytes, *10 9 /l 0.99 ± 0.57 0.91 ± 0.45 0.630 CD4 + T cells, counts/ml 472.1 ± 407 464.1 ± 441.3 0.967 Hemoglobin, g/l 99.1 ± 22.6 100.8 ± 23.6 0.817 Blood platelet count, *10 9 /l 252.7 ± 132.9 229.7 ± 121.7 0.568 Serum albumin, g/l 32.2 ± 4.3 31.2 ± 3.6 0.438 LDH, IU/l 279.3 ± 111.9 248.8 ± 132.8 0.417 PaO2/FIO2 237.2 ± 155.5 210.7 ± 134.9 0.977 CRB-65, 0/1/2/3/4 7/11/5/2/2 4/6/3/3/1 0.633 prednisolone (equivalent) dose, mg/day 36.1 ± 18.4 29.0 ± 26.4 0.296 CTX/RTX/AZA/none, n 4/4/3/15 2/1/3/11 0.772 Abbreviation: AZA, azathioprine; BALF, bronchoalveolar lavage fluid; CMT, conventional microbiological tests; CRP, C-reactive protein; CTX, cyclophosphamide; eGFR, estimated glomerular filtration rate; ESR erythrocyte sedimentation rate; LDH, lactic dehydrogenase; mNGS, metagenomic next-generation sequencing; RTX, rituximab; WBC, white blood cell counts in peripheral blood The results of microbiological tests in the BALF mNGS group The results of microbiological tests in the 27 patients in the BALF mNGS group are described in Table 2 . A total of 25.9% (7/27) of the patients did not have any evidence of pathogenic microorganism in their BALF samples according to BALF mNGS tests, while 40.7% (11/27) of the patients did not have any pathogenic microorganism in their BALF and serum samples according to CMTs tests. At the same time, a total of 55.6% (15/27) of patients had polymicrobial infections, including bacteria, fungi and viruses, according to both CMTs and BALF mNGS tests. Most results of BALF mNGS tests were consistent with those of CMTs, but BALF mNGS detected more pathogens than CMTs, as the positive rate was significantly higher according to BALF mNGS tests than CMTs tests ( P = 0.045). Subsequent treatment adjustments and outcomes are also summarized in Table 2 . Table 2 The results of microbiological tests in patients in the BALF mNGS group Patient No. Sex/Age Conventional microbiological tests mNGS tests Symptomatic infection duration before bronchoscopies, days Teatment before bronchoscopies Treatment adjustment upon mNGS tests Outcomes 1 M/80 Fungal spores (sputum culture)/ BALF GM(+)/hMPV (throat swab PCR) Aspergillus fumigatus 12 PE, prednisolone, antibiotics voriconazole, prednisolone withdrawal death 2 M/63 none none 0 - plasmapheresis, prednisolone, RTX survival 3 M/66 Serum G(+)/Pneumocystis cysts (BALF smear)/RhV (throat swab PCR) Pneumocystis jirovecii/RhV-A 4 PE, MP pulse, prednisolone, RTX, antibiotics SMZ/TMP, prednisolone withdrawal survival 4 F/66 Serum G(+)/Pneumocystis cysts (BALF smear)/BALF GM(+)/BALF CMV-DNA(+) Bca/Hin/Aspergillus fumigatus/Pneumocystis jirovecii/CMV/HHV-7 5 MP pulse, prednisolone, RTX, antibiotics antibiotics upgrading, voriconazole, SMZ-TMP, prednisolone withdrawal survival 5 M/65 none Sps/CMV 6 MP pulse, prednisolone AZA antibiotics, immunosuppressive therapy continuance survival 6 F/48 none none 4 PE, prednisolone, RTX, antibiotics antibiotics discontinuance survival 7 F/57 Pae (BALF culture) Pae/Sep 9 prednisolone, AZA antibiotics, immunosuppressive therapy continuance survival 8 F/65 Serum G(+)/Pneumocystis cysts (BALF smear) Pneumocystis jirovecii/P. chrysogenum species complex/CMV/HHV-6B 6 prednisolone, CTX, antibiotics SMZ-TMP, ganciclovir, prednisolone withdrawal death 9 M/71 none Pae/Nocardia pneumoniae /Candida albicans 4 prednisolone, AZA antibiotics, SMZ-TMP, immunosuppressive therapy continuance survival 10 F/67 none none 1 prednisolone, antibiotics antibiotics discontinuance, immunosuppressive therapy intensification survival 11 F/65 Candida albicans (sputum culture) Sho/ HCov-OC43/ Candida albicans 9 prednisolone, CTX, antibiotics antibiotics upgrading, prednisolone withdrawal death 12 F/71 Pae (sputum and BALF culture) Pae 13 PE, MP pulse, prednisolone, CTX antibiotics, prednisolone withdrawal survival 13 M/58 Serum G(+) Pneumocystis jirovecii 4 MP pulse, prednisolone, CTX, antibiotics SMZ-TMP, prednisolone withdrawal survival 14 F/60 Serum G(+)/BALF CMV-DNA(+) Efa/ Pneumocystis jirovecii/CMV/EBV 4 prednisolone, antibiotics antibiotics upgrading, SMZ-TMP, prednisolone withdrawal survivalESRD 15 M/61 none Pae 8 prednisolone antibiotics, PE, immunosuppressive therapy continuance survival 16 M/70 BALF EBV-DNA(+) Sps/EBV/ Candida albicans 0 - prednisolone, CTX, antibiotics survival 17 F/77 none none 1 antibiotics antibiotics discontinuance prednisolone, CTX survival 18 M/67 Pae (sputum and BALF culture) Pae/ Achromobacter xylosoxidans/RhV-A 9 prednisolone, antibiotics antibiotics upgrading, prednisolone withdrawal survivalESRD 19 M/82 BALF CMV-DNA(+) C. amycolatum 0 prednisolone, ganciclovir antibiotics, immunosuppressive therapy continuance survival 20 F/54 Candida tropicalis (sputum culture) Bca/C. striatum/ Candida albicans/ Candida tropicalis 5 PE, prednisolone antibiotics, prednisolone withdrawal survival 21 F/76 none none 3 prednisolone immunosuppressive therapy intensification survival 22 F/44 none none 4 prednisolone immunosuppressive therapy intensification survival 23 M/71 BALF CMV-DNA(+) Efa/Sho /HCov/EBV/CMV/Candida parapsilosis 7 PE, prednisolone, antibiotics antibiotics adjustment, ganciclovir, prednisolone withdrawal survivalESRD 24 F/59 none Sau/Sma 4 prednisolone, antibiotics antibiotics upgrading, prednisolone withdrawal survival 25 F/75 Serum PIV IgM (+)/Pneumocystis cysts (BALF smear)/BALF EBV-DNA(+) Pneumocystis jirovecii/ EBV/CMV/RSV 0 prednisolone, CTX SMZ-TMP, ganciclovir, antibiotics, prednisolone withdrawal survival 26 F/75 none none 2 antibiotics, prednisolone antibiotics discontinuance, immunosuppressive therapy intensification survival 27 F/72 Sho (BALF culture)/ Serum G(+)/BALF GM(+) Sho/Kpn/Pneumocystis jirovecii/ Aspergillus terreus EBV/CMV 0 fluconazole, SMZ-TMP, antibiotics, prednisolone voriconazole, antibiotics adjustment, prednisolone withdrawal survival Abbreviation: AZA, azathioprine; Bca, Moraxella catarrhalis; C. amycolatum, Corynebacterium amycolatum; CMV, cytomegalovirus; C. striatum, Corynebacterium striatum; CTX, cyclophosphamide; EBV, human gammaherpesvirus 4; Efa, Enterococcus faecium; ESRD, end-stage renal disease; HCov, human coronavirus; HHV, human herpesvirus; Hin, Haemophilus influenza; hMPV, human metapneumovirus; Kpn, Klebsiella pneumoniae; MP, methylprednisolone; mNGS, metagenomic next-generation sequencing; Pae, Pseudomonas aeruginosa; P. chrysogenum, Penicillium chrysogenum; PE, plasmapheresis; PIV, parainfluenza virus; RhV-A, rhinovirus A; RSV, respiratory syncytial virus; RTX, rituximab; Sau, Staphylococcus aureus; Sep, Staphylococcus epidermidis; Sho, Staphylococcus hominis; Sma, Stenotrophomonas maltophilia; SMZ-TMP, sulfamethoxazole and trimethoprim; Sps, Streptococcus pseudopneumoniae; Shorter duration of antimicrobial treatment in the BALF mNGS group The durations of antibacterial, antifungal, antiviral and total antimicrobial treatment in patients were compared between the BALF mNGS group and the CMT alone group. Patients in the BALF mNGS group received significantly shorter duration of antibacterial and total antimicrobial treatment than patients in the CMT alone group (17.3 ± 14.7 vs. 27.9 ± 19.0 days, P = 0.044; 18.9 ± 15.0 vs. 29.5 ± 17.7 days, P = 0.040, respectively), while patients in the BALF mNGS group received comparable duration of antifungal and antiviral treatment to patients in the CMT alone group (11.7 ± 12.6 vs. 9.4 ± 17.4 days, P = 0.607; 3.2 ± 6.8 vs. 1.5 ± 5.2 days, P = 0.382, respectively) (Fig. 1 ). We then divided patients in the BALF mNGS group into two subgroups based on the median duration from occurrence of symptoms of suspected infection to bronchoscopies, i.e., < 5 days and ≥ 5 days, termed the early BALF mNGS group and late BALF mNGS group, respectively. Patients in the early BALF mNGS group received significantly shorter antibacterial and total antimicrobial treatment than patients in the late BALF mNGS group (11.3 ± 11.4 vs. 26.1 ± 14.8 days, P = 0.007; 13.3 ± 12.9 vs. 27.1 ± 14.5 days, P = 0.016, respectively). Lower in-hospital mortality in the BALF mNGS group The in-hospital mortality, ICU stay and duration of ventilation between the BALF mNGS group and the CMT alone group were compared. Patients in the BALF mNGS group had significantly lower in-hospital mortality than patients in the CMT alone group (4/27 vs. 7/17, P = 0.049). All the causes of mortality were severe infections. However, all the 7 patients without any pathogenic microorganisms in their BALF samples according to BALF mNGS tests received intensive immunosuppressive therapy, and none of them developed infection during hospitalization after the treatment. There was no significant difference in the duration of ICU stay or ventilation in patients between these two groups. However, no deaths occurred in the early BALF mNGS group, defined as duration from occurrence of symptoms of suspected infection to bronchoscopies < 5 days (Table 2 ). DISCUSSION In the current study, we summarized the application of BALF mNGS tests in 27 active AAV patients who received immunosuppressive therapy and were suspected of pulmonary infection. We also compared the duration of antimicrobial treatment, as well as in-hospital mortality between these 27 patients and another 17 active AAV patients, diagnosed during the same period, whose samples were only tested by conventional microbiological tests. We found that the duration of antibacterial and total antimicrobial treatment was significantly shorter in patients whose BALF samples were available for mNGS tests. Due to precise antimicrobial treatment, a significantly lower proportion of patients died of severe infection in the BALF mNGS group, as compared with the CMT alone group. Moreover, patients would benefit more from early mNGS tests. To the best of our knowledge, this is the first report of applying mNGS in the management of pulmonary infection in patients with AAV. According to our results, the mNGS test could generate benefits as a rule-out test for infection. By weighing the benefits and limitations of mNGS, Blauwkamp found that immunocompromised patients presenting with non-specific symptoms of infection could potentially benefit from this approach including rule-out testing for sepsis when the standard of care was too invasive or was not sensitive enough ( 25 ). Furthermore, Qian’s study showed that the negative prediction accuracy rate was convincing when the mNGS result was negative. The authors also suggested that if there are indications of respiratory tract infection, respiratory tract samples were recommended for the mNGS test because the negative rates of sputum and BALF were much lower than other types of samples ( 26 ). Early identification of infecting pathogens is crucial for targeted and appropriate antimicrobial therapy, but physicians typically prescribe antimicrobials empirically, based on clinical guidelines and local epidemiology, before microbiological test results are available. The mNGS test could provide more useful information for adjusting antimicrobial treatment, as we found that the duration of antibacterial and total antimicrobial treatment was significantly shorter in patients whose BALF samples were timely available for mNGS tests. First, mNGS test has advantages in turnaround time over conventional microbiological tests ( 27 , 28 ). Although microbiological culture is the standard diagnostic test for many pathogens, it is also time-consuming for the turnaround time to results of ≥ 2 days, and some pathogens are difficult to culture. Second, consistent with other immunocompromised patients with pulmonary infection ( 18 ), most AAV patients under immunosuppressive therapy with pulmonary infection had atypical polymicrobial infection, including bacterium, fungus and virus. As a relatively sensitive method with high detection rates, mNGS tests could provide evidence for adequate and precise antimicrobial treatment. Last but not least, mNGS tests could provide evidence for adjusting treatment strategy in time for physicians. For instance, Patient No.4 (Table 2 ) in the current received a second fiberoptic bronchoscopy and BALF mNGS test two weeks later when his condition worsened despite aggressive antimicrobial treatment. However, the results showed a much lower incidence of the pathogen RPTM, so the treatment strategy was adjusted from intensive antimicrobial treatment to enhanced immunosuppressive treatment. In this way, it was helpful to avoid unnecessary side-effects of antimicrobial treatment and overuse of antibiotics. In conclusion, compared with conventional microbiological tests, using mNGS tests may facilitate shortening and decreasing antimicrobial treatment, and decreasing mortality of severe infection. Therefore, we recommended mNGS as a useful rule-out tool in differentiating pulmonary infection from pulmonary involvement of vasculitis in AAV patients, and mNGS may offer the potential for early implementation of accurate therapy in AAV patients with suspected pulmonary infection. Declarations Acknowledgments Not applicable. Authors’ contributions Chen Wang and Zhi-Ying Li designed and planned the study. Chen wang handled the selection of suitable patients for the study, arranged the collection of clinical data, performed data analysis and drafted the manuscript. Zhan-Wei Hu conducted patients’ bronchoscopies. Zhi-Ying Li contributed to manuscript preparation. Ming-Hui Zhao and Min Chen guided the study as senior authors. All the authors contributed to data interpretation and manuscript revision. The author(s) read and approved the final manuscript. Funding This study was supported by three grants from the National Natural Science Fund (Nos. 81870477, 81870478 and 82090020/82090021), a grant from the Capital’s Funds for Health Improvement and Research (No. 2020-2-4073), a grant from Chinese Academy of Medical Sciences Research Unit (No. 2019RU023), a grant from National High Level Hospital Clinical Research Funding (Multi-center Clinical Research Project of Peking University First Hospital [No. 2022CR52]) and CAMS Innovation Fund for Medical Science (No. 2019-I2M-5-046). Availability of data and materials Data collection has been conducted in accordance to local regulation. The authors declare their availability in providing data if requested by the referees or the editorial team of the journal. All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author. Ethics approval and consent to participate The research was in compliance with the Declaration of Helsinki and was approved by the clinical research ethics committee of the Peking University First Hospital. Written informed consent was obtained from the patients or their guardians. Consent for publication All authors have read the final draft of the article and approved its submission for publication. Competing interests The authors declare that they have no competing interests. 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Predictors for outcomes in patients with severe ANCA-associated glomerulonephritis who were dialysis-dependent at presentation: a study of 89 cases in a single Chinese center. Semin Arthritis Rheum. 2013;42(5):515–21. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF 3rd, Feldman HI, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009;150(9):604–12. Edwards L, Perrin K, Wijesinghe M, Weatherall M, Beasley R, Travers J. The value of the CRB65 score to predict mortality in exacerbations of COPD requiring hospital admission. Respirology. 2011;16(4):625–9. Patterson TF, Thompson GR 3rd, Denning DW, Fishman JA, Hadley S, Herbrecht R, et al. Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America. Clin Infect Dis. 2016;63(4):e1–60. Lee HY, Rhee CK, Choi JY, Lee HY, Lee JW, Lee DG. Diagnosis of cytomegalovirus pneumonia by quantitative polymerase chain reaction using bronchial washing fluid from patients with hematologic malignancies. Oncotarget. 2017;8(24):39736–45. Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol. 2019;4(4):663–74. Qian M, Zhu B, Zhan Y, Wang L, Shen Q, Zhang M, et al. Analysis of Negative Results of Metagenomics Next-Generation Sequencing in Clinical Practice. Front Cell Infect Microbiol. 2022;12:892076. Ju CR, Lian QY, Guan WJ, Chen A, Zhang JH, Xu X, et al. Metagenomic Next-Generation Sequencing for Diagnosing Infections in Lung Transplant Recipients: A Retrospective Study. Transpl Int. 2022;35:10265. Parize P, Muth E, Richaud C, Gratigny M, Pilmis B, Lamamy A, et al. Untargeted next-generation sequencing-based first-line diagnosis of infection in immunocompromised adults: a multicentre, blinded, prospective study. Clin Microbiol Infect. 2017;23(8):574. .e1-.e6 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 27 Sep, 2024 Read the published version in BMC Pulmonary Medicine → Version 1 posted Editorial decision: Revision requested 12 Aug, 2024 Reviews received at journal 09 Aug, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers agreed at journal 14 Jun, 2024 Reviews received at journal 25 Apr, 2024 Reviewers agreed at journal 17 Apr, 2024 Reviewers agreed at journal 17 Apr, 2024 Reviewers invited by journal 15 Apr, 2024 Editor assigned by journal 15 Apr, 2024 Editor invited by journal 18 Mar, 2024 Submission checks completed at journal 18 Mar, 2024 First submitted to journal 25 Feb, 2024 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-3989390","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280736363,"identity":"d4300c97-6e69-4269-9aaf-c13ad4a9ee51","order_by":0,"name":"Chen Wang","email":"","orcid":"","institution":"Peking University First Hospital, Peking University Institute of Nephrology","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Wang","suffix":""},{"id":280736364,"identity":"2c7d3c2a-9876-48a1-8d84-230ce9386c17","order_by":1,"name":"Zhan-Wei Hu","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhan-Wei","middleName":"","lastName":"Hu","suffix":""},{"id":280736365,"identity":"da63920f-c3a8-45e2-aeb9-e4026e9e1088","order_by":2,"name":"Zhi-Ying Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACNmbmAwcSDP7L8fM3H2OQAIsl4NfCz96WeOBDBbOx5IxjacRpkew5o3xwxhnmRIMDOWZQMQJaDG7kMBzmbWNLkGw48+2BZc5hoL05Bgw/d+DTknsAqIUnj5+5d7uB5LbDQHvfGDD2nsGnJS8BqEWiWLLh7DYJkBagvQbMjG14HWYA1GKQuOFAzjOwFntCWoDeNwB6PwGkhQ1iiwQBLcBATgAG8gFQIJsD/ZLOI3HmWcHBXjxagFF5+EOCwQFQVD57LLnNWo6/PXnjg594tKAAZmBU8oAYB4jUwMDA+IFopaNgFIyCUTCSAADbx1qLi4SmngAAAABJRU5ErkJggg==","orcid":"","institution":"Peking University First Hospital, Peking University Institute of Nephrology","correspondingAuthor":true,"prefix":"","firstName":"Zhi-Ying","middleName":"","lastName":"Li","suffix":""},{"id":280736368,"identity":"278630dd-148f-4159-91ac-73738c4190ed","order_by":3,"name":"Ming-Hui Zhao","email":"","orcid":"","institution":"Peking University First Hospital, Peking University Institute of Nephrology","correspondingAuthor":false,"prefix":"","firstName":"Ming-Hui","middleName":"","lastName":"Zhao","suffix":""},{"id":280736372,"identity":"8ee4b09b-7326-41cf-9261-94996035caca","order_by":4,"name":"Min Chen","email":"","orcid":"","institution":"Peking University First Hospital, Peking University Institute of Nephrology","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-02-26 00:45:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3989390/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3989390/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12890-024-03301-5","type":"published","date":"2024-09-27T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53193220,"identity":"01622fe4-ebbb-4185-9cd9-886552c21834","added_by":"auto","created_at":"2024-03-21 18:00:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":183843,"visible":true,"origin":"","legend":"\u003cp\u003eShorter duration of antimicrobial treatment in the BALF mNGS group\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3989390/v1/0fc7b9264353b6a7149b5ae2.jpeg"},{"id":65627206,"identity":"7a8e0baf-1ece-46f3-88a5-6b8aea8300f6","added_by":"auto","created_at":"2024-09-30 16:13:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":891072,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3989390/v1/bfb1e180-5b1a-4dd7-938f-04915af99bc8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Advantages of metagenomic next-generation sequencing in the management of ANCA-associated vasculitis patients with pulmonary infections as a rule-out tool","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAntineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) comprises granulomatosis with polyangiitis (GPA, previously known as Wegener\u0026rsquo;s granulomatosis), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA, previously known as Churg-Strauss syndrome) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The lung and kidney are the two most involved organs in AAV. Pulmonary involvement occurs in 25\u0026ndash;80% of AAV patients (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), including interstitial lung disease, cavity, nodules, and alveolar hemorrhage, and diffuse alveolar hemorrhage (DAH) is one of the most severe manifestations of AAV (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), significantly contributing to morbidity and mortality.\u003c/p\u003e \u003cp\u003eAAV is usually fatal when left untreated, with a 1-year mortality rate of approximately 80% (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Although the introduction of immunosuppressive therapy substantially improved the outcome of AAV, it was also associated with high rates of adverse events, especially secondary infection (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Consistent with studies from other groups (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), we found that secondary infection was the leading cause of death during the first year after the diagnosis of AAV (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and the majority of infectious complication was pulmonary infection (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Therefore, early recognition and appropriate treatment in time are of great importance.\u003c/p\u003e \u003cp\u003eHowever, it is sometimes difficult to differentiate pulmonary infection from pulmonary involvement of vasculitis in AAV patients due to similar clinical manifestations (eg. fever, cough and hemoptysis), laboratory findings (eg. elevated white blood cell and neutrophil counts in peripheral blood, C-reactive protein [CRP] and erythrocyte sedimentation rate [ESR]) and imaging features (patches, masses, ground-glass opacities and consolidations). Moreover, given that the treatment strategies for pulmonary involvement of active AAV and pulmonary infection are inconsistent, the hard differential diagnosis presents us with difficulties in treatment decisions. Misdiagnosing pulmonary infection as active AAV pulmonary involvement and thus treating with aggressive immunosuppressive therapy could probably result in disastrous consequences of aggravation of infection. Fiberoptic bronchoscopy and bronchoalveolar lavage fluid (BALF) assays are useful diagnostic methods when a co-existing infection is suspected in AAV patients with pulmonary involvement. In addition to conventional microbiological tests (CMTs) of BALF, metagenomic next-generation sequencing (mNGS) is a nucleic acid sequencing technique with a high-throughput capacity and an approach that can theoretically detect any microorganism in the bio-sample, including BALF (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Various studies have reported the application of mNGS to detect pathogens in pulmonary infection (\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In the study by Peng et al., which involved immunocompromised patients with pulmonary infection, it was found that the diagnostic performance of BALF mNGS was similar to that of comprehensive CMTs, but when there is a lack of consideration of potential pathogens, the combination of mNGS and CMTs might be a better diagnostic strategy (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo our knowledge, there is few, if any, reports on the application of mNGS in AAV. Thus the current study evaluated the advantages of additional BALF mNGS in the management of pulmonary infection in patients with AAV.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003e AAV patients diagnosed at Peking University First Hospital between March 2019 and March 2022 were reviewed. Treatment protocols were described previously (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In brief, patients received the induction therapy typically including corticosteroids in combination with cyclophosphamide (CTX) or rituximab (RTX). Patients with severe pulmonary hemorrhage or acute renal failure requiring dialysis at diagnosis received additional methylprednisolone pulse and plasmapheresis. For maintenance therapy, daily oral azathioprine (AZA) or RTX was administered. Those who were clinically suspected of pulmonary infection and underwent fiberoptic bronchoscopy were retrospectively recruited. All patients met the Chapel Hill Consensus Conference criteria for AAV (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Patients with secondary vasculitis or with other comorbid renal diseases were excluded. 27 patients whose BALF samples were simultaneously available for mNGS tests besides CMTs (termed the BALF mNGS group), and 17 patients whose BALF was only tested by CMTs (termed the CMT alone group), were included in the study. This research was conducted in accordance with the Declaration of Helsinki and was approved by the Clinical Research Ethics Committee of the Peking University First Hospital. Written informed consent was obtained from the patients or their guardians.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eData collection\u003c/h3\u003e\n\u003cp\u003eAll the clinical and laboratory data were collected from the medical records of the patients at admission and at the time of bronchoscopies, including age, sex, vital signs, diagnosis, ANCA serotype, disease phenotype, organ involvement, laboratory data (serum creatinine, CRP, ESR, white blood cell [WBC], neutrophil and lymphocyte counts, hemoglobin, platelet count, CD4\u0026thinsp;+\u0026thinsp;T cells, serum albumin, lactate dehydrogenase [LDH] and arterial blood gas analysis), immunosuppressive therapies, antimicrobial treatment, concomitant treatment, respiratory support, response to treatment and intensive care unit (ICU) stay. The estimated glomerular filtration rate (eGFR) was calculated by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) Eq.\u0026nbsp;(21). The risk scores CRB65 were calculated based on the initial available data, which involve mental status, respiratory rate, blood pressure and age (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSpecimen collection\u003c/h2\u003e \u003cp\u003eAll bronchoscopies were performed by experienced bronchoscopists. The target lesion was decided according to the chest CT beforehand. A standard bronchoalveolar lavage was collected before any other bronchoscopy procedures to avoid possible contamination. Once collected, BALF specimens were immediately sent to the clinical microbiology laboratory for CMTs and to a commercial laboratory for mNGS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eConventional microbiological tests\u003c/h2\u003e \u003cp\u003eComprehensive CMTs were performed on BALF, serum, sputum and throat swab samples, including culture, antigen detection, multiplex PCR, and direct microscopic examination of specimens according to standard procedures (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Gram staining, acid-fast staining, hexamine silver staining, the galactomannan antigen detection test (GM test), cryptococcal capsular polysaccharide antigen test and \u003cem\u003eAspergillus\u003c/em\u003e antibody detection with BALF samples, interferon-gamma release assay (T-SPOT. TB), (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)-β-D-glucan test (G test) and GM test with serum samples were performed to identify bacteria, including \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e, and fungi, including \u003cem\u003ePneumocystis carinii\u003c/em\u003e, \u003cem\u003eCryptococcus neoformans, Candida\u003c/em\u003e and \u003cem\u003eAspergillus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003emNGS of BALF\u003c/h2\u003e \u003cp\u003emNGS was performed once the sample was obtained. The total turnaround time is approximately 24 hours (5 hours for shipping and 19 hours for testing). BALF samples were collected from patients for metagenomic analysis pipeline for microbial identification (MAPMI). Total DNA and RNA were extracted from clinical samples by using a MAPMI sample preparation kit (CapitalBio Technology, Beijing, China. Catalogue No. S60150) according to the manufacturer\u0026rsquo;s instructions. The extracted RNA was reverse-transcribed using random primers, and cDNA was pooled with DNA from the same clinical sample for sequencing library preparation. The pooled nucleic acid was enzymatically fragmented to a size of 200\u0026ndash;300 base pairs (bp), and sequencing libraries were constructed through end repair, adapter ligation and PCR amplification. Sequencing templates were further prepared with the OneTouch2 System (Life Technologies, CA, USA) and sequenced on the BioelectronSeq 4000 sequencer (CapitalBio Technology) after quality control. A negative control sample of water was used in each run to monitor potential contamination. The original sequencing data were subjected to quality control, and reads with lengths less than 50bp, low quality, or low complexity were removed. The remaining high-quality sequencing data were mapped to the human reference genome grch38 for the depletion of human host sequences using Bowtie2 software. Subsequently, the nonhuman sequences were classified by simultaneous alignment to the genomic sequence databases downloaded from the NCBI and PATRIC databases. To judge the suspected pathogens in clinical samples, we reviewed data from different types of samples from healthy people and calculated the relevant reference values, including the hit read number and coverage of all bacteria, fungi, viruses and parasites detected. Moreover, pathogens detected in the negative control sample of water were removed from the results of clinical samples. The final pathogen detection results included a list of suspected pathogens, the number of hit reads (using the term reads per ten million [RPTM]) and genome-level coverage statistics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (for data that were normally distributed) or median and interquartile range (IQR; for data that were in skewed distribution) for continuous variables and number (%) for categorical variables as appropriate. Quantitative parameters were compared using Student\u0026rsquo;s t test (for data that were normally distributed) or the nonparametric test (for data that were in skewed distribution) as appropriate. Categorical variables were compared using the \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e test. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analysis was performed using SPSS 18.0 (Chicago, IL, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGeneral data of the AAV patients\u003c/h2\u003e \u003cp\u003eThe clinical and laboratory data of the patients in the BALF mNGS group and CMT alone group are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. No significant difference in age, sex, laboratory data, the ratio of partial pressure to fraction of inspiration oxygen (PaO2/FiO2), CRB-65, or immunosuppressive regimens was found between these two groups of patients. Therefore, there was no significant difference in disease activity of AAV, severity of infection or intensity of immunosuppressive therapy between the two groups.\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\u003eGeneral data of the patients in the BALF mNGS group and CMT alone group\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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBALF mNGS group (n\u0026thinsp;=\u0026thinsp;27)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCMTs group\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e values\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\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.6\u0026thinsp;\u0026plusmn;\u0026thinsp;18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.371\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(41%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (65%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.126\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMPO-ANCA/PR3-ANCA, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.786\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eANCA titers, AU/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e122.8\u0026thinsp;\u0026plusmn;\u0026thinsp;111.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108.3\u0026thinsp;\u0026plusmn;\u0026thinsp;87.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.657\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum creatinine, umol/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e329.4\u0026thinsp;\u0026plusmn;\u0026thinsp;232.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e342.7\u0026thinsp;\u0026plusmn;\u0026thinsp;249.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.858\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eeGFR, ml/min/1.73m\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.2\u0026thinsp;\u0026plusmn;\u0026thinsp;31.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.3\u0026thinsp;\u0026plusmn;\u0026thinsp;28.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.995\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRP, mg/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.6 (13.5/90.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.0 (5.0/107.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.990\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eESR, mm/1h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.1\u0026thinsp;\u0026plusmn;\u0026thinsp;44.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.3\u0026thinsp;\u0026plusmn;\u0026thinsp;33.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.642\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCT, ng/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.34 (0.13/1.52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.37 (0.14/1.34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.951\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC, *10\u003csup\u003e9\u003c/sup\u003e/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.292\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophils, *10\u003csup\u003e9\u003c/sup\u003e/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.256\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocytes, *10\u003csup\u003e9\u003c/sup\u003e/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.630\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD4\u0026thinsp;+\u0026thinsp;T cells, counts/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e472.1\u0026thinsp;\u0026plusmn;\u0026thinsp;407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e464.1\u0026thinsp;\u0026plusmn;\u0026thinsp;441.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.967\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin, g/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.1\u0026thinsp;\u0026plusmn;\u0026thinsp;22.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.8\u0026thinsp;\u0026plusmn;\u0026thinsp;23.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.817\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood platelet count, *10\u003csup\u003e9\u003c/sup\u003e/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e252.7\u0026thinsp;\u0026plusmn;\u0026thinsp;132.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e229.7\u0026thinsp;\u0026plusmn;\u0026thinsp;121.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.568\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum albumin, g/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.438\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLDH, IU/l\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e279.3\u0026thinsp;\u0026plusmn;\u0026thinsp;111.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e248.8\u0026thinsp;\u0026plusmn;\u0026thinsp;132.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.417\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePaO2/FIO2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e237.2\u0026thinsp;\u0026plusmn;\u0026thinsp;155.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e210.7\u0026thinsp;\u0026plusmn;\u0026thinsp;134.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.977\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRB-65, 0/1/2/3/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/11/5/2/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4/6/3/3/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.633\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eprednisolone (equivalent) dose, mg/day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;18.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.0\u0026thinsp;\u0026plusmn;\u0026thinsp;26.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.296\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCTX/RTX/AZA/none, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4/4/3/15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2/1/3/11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.772\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviation: AZA, azathioprine; BALF, bronchoalveolar lavage fluid; CMT, conventional microbiological tests; CRP, C-reactive protein; CTX, cyclophosphamide; eGFR, estimated glomerular filtration rate; ESR erythrocyte sedimentation rate; LDH, lactic dehydrogenase; mNGS, metagenomic next-generation sequencing; RTX, rituximab; WBC, white blood cell counts in peripheral blood\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe results of microbiological tests in the BALF mNGS group\u003c/h2\u003e \u003cp\u003eThe results of microbiological tests in the 27 patients in the BALF mNGS group are described in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A total of 25.9% (7/27) of the patients did not have any evidence of pathogenic microorganism in their BALF samples according to BALF mNGS tests, while 40.7% (11/27) of the patients did not have any pathogenic microorganism in their BALF and serum samples according to CMTs tests. At the same time, a total of 55.6% (15/27) of patients had polymicrobial infections, including bacteria, fungi and viruses, according to both CMTs and BALF mNGS tests. Most results of BALF mNGS tests were consistent with those of CMTs, but BALF mNGS detected more pathogens than CMTs, as the positive rate was significantly higher according to BALF mNGS tests than CMTs tests (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045). Subsequent treatment adjustments and outcomes are also summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\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\u003eThe results of microbiological tests in patients in the BALF mNGS group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\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\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSex/Age\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConventional microbiological tests\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003emNGS tests\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eSymptomatic infection duration before bronchoscopies, days\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTeatment before bronchoscopies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eTreatment adjustment upon mNGS tests\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eOutcomes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM/80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eFungal spores (sputum culture)/ BALF GM(+)/hMPV (throat swab\u0026nbsp;PCR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eAspergillus fumigatus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePE, prednisolone, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003evoriconazole, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003edeath\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eplasmapheresis, prednisolone, RTX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSerum G(+)/Pneumocystis cysts (BALF smear)/RhV (throat swab\u0026nbsp;PCR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePneumocystis jirovecii/RhV-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePE,\u003c/p\u003e \u003cp\u003eMP pulse, prednisolone, RTX, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSMZ/TMP, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSerum G(+)/Pneumocystis cysts (BALF smear)/BALF GM(+)/BALF CMV-DNA(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eBca/Hin/Aspergillus fumigatus/Pneumocystis jirovecii/CMV/HHV-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMP pulse, prednisolone, RTX, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics upgrading, voriconazole, SMZ-TMP, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSps/CMV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMP pulse, prednisolone\u003c/p\u003e \u003cp\u003eAZA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics, immunosuppressive therapy continuance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePE, prednisolone, RTX, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics discontinuance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ePae (BALF culture)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePae/Sep\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, AZA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics, immunosuppressive therapy continuance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSerum G(+)/Pneumocystis cysts (BALF smear)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePneumocystis jirovecii/P. chrysogenum species complex/CMV/HHV-6B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, CTX, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSMZ-TMP, ganciclovir, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003edeath\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePae/Nocardia pneumoniae /Candida albicans\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, AZA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics,\u003c/p\u003e \u003cp\u003eSMZ-TMP, immunosuppressive therapy continuance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics discontinuance, immunosuppressive therapy intensification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCandida albicans (sputum culture)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSho/ HCov-OC43/ Candida albicans\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, CTX, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics upgrading, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003edeath\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ePae (sputum and BALF culture)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePE,\u003c/p\u003e \u003cp\u003eMP pulse,\u003c/p\u003e \u003cp\u003eprednisolone, CTX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSerum G(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePneumocystis jirovecii\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMP pulse, prednisolone, CTX, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSMZ-TMP, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSerum G(+)/BALF CMV-DNA(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eEfa/ Pneumocystis jirovecii/CMV/EBV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics upgrading, SMZ-TMP, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvivalESRD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics,\u003c/p\u003e \u003cp\u003ePE, immunosuppressive therapy continuance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBALF EBV-DNA(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSps/EBV/ Candida albicans\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eprednisolone, CTX,\u003c/p\u003e \u003cp\u003eantibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eantibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics discontinuance prednisolone, CTX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ePae (sputum and BALF culture)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePae/ Achromobacter xylosoxidans/RhV-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics upgrading, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvivalESRD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBALF CMV-DNA(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eC. amycolatum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone,\u003c/p\u003e \u003cp\u003eganciclovir\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics, immunosuppressive therapy continuance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCandida tropicalis (sputum culture)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eBca/C. striatum/ Candida albicans/ Candida tropicalis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePE,\u003c/p\u003e \u003cp\u003eprednisolone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eimmunosuppressive therapy intensification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eimmunosuppressive therapy intensification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM/71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBALF CMV-DNA(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eEfa/Sho /HCov/EBV/CMV/Candida parapsilosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePE,\u003c/p\u003e \u003cp\u003eprednisolone, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics adjustment, ganciclovir,\u003c/p\u003e \u003cp\u003eprednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvivalESRD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSau/Sma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, antibiotics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics upgrading, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSerum PIV IgM (+)/Pneumocystis cysts (BALF smear)/BALF EBV-DNA(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ePneumocystis jirovecii/ EBV/CMV/RSV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eprednisolone, CTX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSMZ-TMP, ganciclovir, antibiotics, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eantibiotics, prednisolone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eantibiotics discontinuance, immunosuppressive therapy intensification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSho (BALF culture)/ Serum G(+)/BALF GM(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSho/Kpn/Pneumocystis jirovecii/ Aspergillus terreus\u003c/p\u003e \u003cp\u003eEBV/CMV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003efluconazole, SMZ-TMP, antibiotics, prednisolone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003evoriconazole, antibiotics adjustment, prednisolone withdrawal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003esurvival\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"12\"\u003eAbbreviation: AZA, azathioprine; Bca, Moraxella catarrhalis; C. amycolatum, Corynebacterium amycolatum; CMV, cytomegalovirus; C. striatum, Corynebacterium striatum; CTX, cyclophosphamide; EBV, human gammaherpesvirus 4; Efa, Enterococcus faecium; ESRD, end-stage renal disease; HCov, human coronavirus; HHV, human herpesvirus; Hin, Haemophilus influenza; hMPV, human metapneumovirus; Kpn, Klebsiella pneumoniae; MP, methylprednisolone; mNGS, metagenomic next-generation sequencing; Pae, Pseudomonas aeruginosa; P. chrysogenum, Penicillium chrysogenum; PE, plasmapheresis; PIV, parainfluenza virus; RhV-A, rhinovirus A; RSV, respiratory syncytial virus; RTX, rituximab; Sau, Staphylococcus aureus; Sep, Staphylococcus epidermidis; Sho, Staphylococcus hominis; Sma, Stenotrophomonas maltophilia; SMZ-TMP, sulfamethoxazole and trimethoprim; Sps, Streptococcus pseudopneumoniae;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eShorter duration of antimicrobial treatment in the BALF mNGS group\u003c/h2\u003e \u003cp\u003eThe durations of antibacterial, antifungal, antiviral and total antimicrobial treatment in patients were compared between the BALF mNGS group and the CMT alone group. Patients in the BALF mNGS group received significantly shorter duration of antibacterial and total antimicrobial treatment than patients in the CMT alone group (17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;14.7 vs. 27.9\u0026thinsp;\u0026plusmn;\u0026thinsp;19.0 days, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.044; 18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.0 vs. 29.5\u0026thinsp;\u0026plusmn;\u0026thinsp;17.7 days, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040, respectively), while patients in the BALF mNGS group received comparable duration of antifungal and antiviral treatment to patients in the CMT alone group (11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;12.6 vs. 9.4\u0026thinsp;\u0026plusmn;\u0026thinsp;17.4 days, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.607; 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8 vs. 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 days, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.382, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then divided patients in the BALF mNGS group into two subgroups based on the median duration from occurrence of symptoms of suspected infection to bronchoscopies, i.e., \u0026lt;\u0026thinsp;5 days and \u0026ge;\u0026thinsp;5 days, termed the early BALF mNGS group and late BALF mNGS group, respectively. Patients in the early BALF mNGS group received significantly shorter antibacterial and total antimicrobial treatment than patients in the late BALF mNGS group (11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4 vs. 26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;14.8 days, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007; 13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;12.9 vs. 27.1\u0026thinsp;\u0026plusmn;\u0026thinsp;14.5 days, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLower in-hospital mortality in the BALF mNGS group\u003c/h2\u003e \u003cp\u003eThe in-hospital mortality, ICU stay and duration of ventilation between the BALF mNGS group and the CMT alone group were compared. Patients in the BALF mNGS group had significantly lower in-hospital mortality than patients in the CMT alone group (4/27 vs. 7/17, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049). All the causes of mortality were severe infections. However, all the 7 patients without any pathogenic microorganisms in their BALF samples according to BALF mNGS tests received intensive immunosuppressive therapy, and none of them developed infection during hospitalization after the treatment.\u003c/p\u003e \u003cp\u003eThere was no significant difference in the duration of ICU stay or ventilation in patients between these two groups. However, no deaths occurred in the early BALF mNGS group, defined as duration from occurrence of symptoms of suspected infection to bronchoscopies\u0026thinsp;\u0026lt;\u0026thinsp;5 days (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn the current study, we summarized the application of BALF mNGS tests in 27 active AAV patients who received immunosuppressive therapy and were suspected of pulmonary infection. We also compared the duration of antimicrobial treatment, as well as in-hospital mortality between these 27 patients and another 17 active AAV patients, diagnosed during the same period, whose samples were only tested by conventional microbiological tests. We found that the duration of antibacterial and total antimicrobial treatment was significantly shorter in patients whose BALF samples were available for mNGS tests. Due to precise antimicrobial treatment, a significantly lower proportion of patients died of severe infection in the BALF mNGS group, as compared with the CMT alone group. Moreover, patients would benefit more from early mNGS tests. To the best of our knowledge, this is the first report of applying mNGS in the management of pulmonary infection in patients with AAV.\u003c/p\u003e \u003cp\u003eAccording to our results, the mNGS test could generate benefits as a rule-out test for infection. By weighing the benefits and limitations of mNGS, Blauwkamp found that immunocompromised patients presenting with non-specific symptoms of infection could potentially benefit from this approach including rule-out testing for sepsis when the standard of care was too invasive or was not sensitive enough (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Furthermore, Qian\u0026rsquo;s study showed that the negative prediction accuracy rate was convincing when the mNGS result was negative. The authors also suggested that if there are indications of respiratory tract infection, respiratory tract samples were recommended for the mNGS test because the negative rates of sputum and BALF were much lower than other types of samples (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e Early identification of infecting pathogens is crucial for targeted and appropriate antimicrobial therapy, but physicians typically prescribe antimicrobials empirically, based on clinical guidelines and local epidemiology, before microbiological test results are available. The mNGS test could provide more useful information for adjusting antimicrobial treatment, as we found that the duration of antibacterial and total antimicrobial treatment was significantly shorter in patients whose BALF samples were timely available for mNGS tests. First, mNGS test has advantages in turnaround time over conventional microbiological tests (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Although microbiological culture is the standard diagnostic test for many pathogens, it is also time-consuming for the turnaround time to results of \u0026ge;\u0026thinsp;2 days, and some pathogens are difficult to culture. Second, consistent with other immunocompromised patients with pulmonary infection (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), most AAV patients under immunosuppressive therapy with pulmonary infection had atypical polymicrobial infection, including bacterium, fungus and virus. As a relatively sensitive method with high detection rates, mNGS tests could provide evidence for adequate and precise antimicrobial treatment. Last but not least, mNGS tests could provide evidence for adjusting treatment strategy in time for physicians. For instance, Patient No.4 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) in the current received a second fiberoptic bronchoscopy and BALF mNGS test two weeks later when his condition worsened despite aggressive antimicrobial treatment. However, the results showed a much lower incidence of the pathogen RPTM, so the treatment strategy was adjusted from intensive antimicrobial treatment to enhanced immunosuppressive treatment. In this way, it was helpful to avoid unnecessary side-effects of antimicrobial treatment and overuse of antibiotics.\u003c/p\u003e \u003cp\u003eIn conclusion, compared with conventional microbiological tests, using mNGS tests may facilitate shortening and decreasing antimicrobial treatment, and decreasing mortality of severe infection. Therefore, we recommended mNGS as a useful rule-out tool in differentiating pulmonary infection from pulmonary involvement of vasculitis in AAV patients, and mNGS may offer the potential for early implementation of accurate therapy in AAV patients with suspected pulmonary infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChen Wang and Zhi-Ying Li designed and planned the study. Chen wang handled the selection of suitable patients for the study, arranged the collection of clinical data, performed data analysis and drafted the manuscript. Zhan-Wei Hu conducted patients\u0026rsquo; bronchoscopies. Zhi-Ying Li contributed to manuscript preparation. Ming-Hui Zhao and Min Chen guided the study as senior authors. All the authors contributed to data interpretation and manuscript revision. The author(s) read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by three grants from the National Natural Science Fund (Nos. 81870477, 81870478 and 82090020/82090021), a grant from the Capital\u0026rsquo;s Funds for Health Improvement and Research (No. 2020-2-4073), a grant from Chinese Academy of Medical Sciences Research Unit (No. 2019RU023), a grant from National High Level Hospital Clinical Research Funding (Multi-center Clinical Research Project of Peking University First Hospital [No. 2022CR52]) and CAMS Innovation Fund for Medical Science (No. 2019-I2M-5-046).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData collection has been conducted in accordance to local regulation. The authors declare their availability in providing data if requested by the referees or the editorial team of the journal. All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was in compliance with the Declaration of Helsinki and was approved by the clinical research ethics committee of the Peking University First Hospital. Written informed consent was obtained from the patients or their guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read the final draft of the article and approved its submission for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJennette JC, Falk RJ, Bacon PA, Basu N, Cid MC, Ferrario F et al. 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum. 2013;65(1):1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalk RJ, Jennette JC. ANCA small-vessel vasculitis. 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Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children. Clin Infect Dis. 2019;68(11):1847\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Gao H, Meng H, Wang Q, Li S, Chen H, et al. Detection of Pulmonary Infectious Pathogens From Lung Biopsy Tissues by Metagenomic Next-Generation Sequencing. Front Cell Infect Microbiol. 2018;8:205.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng JM, Du B, Qin HY, Wang Q, Shi Y. Metagenomic next-generation sequencing for the diagnosis of suspected pneumonia in immunocompromised patients. J Infect. 2021;82(4):22\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu H, Zhang Y, Yang J, Liu Y, Chen J. Application of mNGS in the Etiological Analysis of Lower Respiratory Tract Infections and the Prediction of Drug Resistance. 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Nat Microbiol. 2019;4(4):663\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian M, Zhu B, Zhan Y, Wang L, Shen Q, Zhang M, et al. Analysis of Negative Results of Metagenomics Next-Generation Sequencing in Clinical Practice. Front Cell Infect Microbiol. 2022;12:892076.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJu CR, Lian QY, Guan WJ, Chen A, Zhang JH, Xu X, et al. Metagenomic Next-Generation Sequencing for Diagnosing Infections in Lung Transplant Recipients: A Retrospective Study. Transpl Int. 2022;35:10265.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParize P, Muth E, Richaud C, Gratigny M, Pilmis B, Lamamy A, et al. Untargeted next-generation sequencing-based first-line diagnosis of infection in immunocompromised adults: a multicentre, blinded, prospective study. Clin Microbiol Infect. 2017;23(8):574. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e.e1-.e6\u003c/span\u003e\u003cspan address=\"http://.e1-.e6\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":"antineutrophil cytoplasmic antibody, vasculitis, pulmonary involvement, pulmonary infection, metagenomic next-generation sequencing","lastPublishedDoi":"10.21203/rs.3.rs-3989390/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3989390/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003ePulmonary infection is one of the leading causes of death in patients with ANCA-associated vasculitis (AAV). It is sometimes difficult to differentiate pulmonary infection from pulmonary involvement of vasculitis in AAV patients. Fiberoptic bronchoscopy and bronchoalveolar lavage fluid (BALF) assays are useful diagnostic methods. In addition to conventional microbiological tests (CMTs), metagenomic next-generation sequencing (mNGS) facilitates rapid and sensitive detection of various pathogens. The current study aimed to evaluate the advantages of additional BALF mNGS in the management of pulmonary infection in AAV patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003e27 patients with active AAV and suspected pulmonaryinfection whose BALF samples were tested by mNGS and CMTs and 17 active AAV patients whose BALF were tested by CMTs alone were retrospectively recruited. The results of microbiological tests and adjustments of treatment upon BALF mNGS were described. The durations of antimicrobial treatment and in-hospital mortality in patients were compared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAmong the 27 patients whose BALF samples were tested by mNGS, 25.9% of patients did not have any evidence of pathogenic microorganism in their BALF samples, and 55.6% of patients had polymicrobial infections, including bacteria, fungi and virus, while 40.7% of patients did not have any evidence of pathogenic microorganism in their BALF and serum samples according to CMTs tests. Patients in the BALF mNGS group received significantly shorter duration of antibacterial and total antimicrobial treatment than patients in the CMT alone group (17.3±14.7 vs. 27.9±19.0 days, \u003cem\u003eP\u003c/em\u003e=0.044; 18.9±15.0 vs. 29.5±17.7 days, \u003cem\u003eP\u003c/em\u003e=0.040, respectively). Patients in the BALF mNGS group had significantly lower in-hospital mortality than patients in the CMT alone group (4/27 vs. 7/17, \u003cem\u003eP\u003c/em\u003e=0.049).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eCompared with CMT alone, additional mNGS tests may shorten the duration of antimicrobial treatment and decrease death from severe infection by providing precise and quick diagnosis of infection.\u003c/p\u003e","manuscriptTitle":"Advantages of metagenomic next-generation sequencing in the management of ANCA-associated vasculitis patients with pulmonary infections as a rule-out tool","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-21 17:59:37","doi":"10.21203/rs.3.rs-3989390/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-12T10:00:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-09T13:07:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180587170332839588641944006782516036976","date":"2024-07-12T14:32:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27119634753962471892751942985897178102","date":"2024-06-14T14:40:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-25T16:15:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9260b8ab-44e8-4b5f-934e-af1c0849d58b","date":"2024-04-17T09:28:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9212d74b-8b25-4481-8a53-97672c6c223e","date":"2024-04-17T08:38:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-15T07:45:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-15T07:31:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-18T07:06:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-18T07:04:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pulmonary Medicine","date":"2024-02-26T00:44:14+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":"775133d1-93a4-466a-a00a-57a3b37fab8e","owner":[],"postedDate":"March 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:02:39+00:00","versionOfRecord":{"articleIdentity":"rs-3989390","link":"https://doi.org/10.1186/s12890-024-03301-5","journal":{"identity":"bmc-pulmonary-medicine","isVorOnly":false,"title":"BMC Pulmonary Medicine"},"publishedOn":"2024-09-27 15:57:28","publishedOnDateReadable":"September 27th, 2024"},"versionCreatedAt":"2024-03-21 17:59:37","video":"","vorDoi":"10.1186/s12890-024-03301-5","vorDoiUrl":"https://doi.org/10.1186/s12890-024-03301-5","workflowStages":[]},"version":"v1","identity":"rs-3989390","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3989390","identity":"rs-3989390","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}