Application Of Metagenomic Next-generation Sequencing In The Diagnosis Of Pulmonary Diseases Of Malignant Hematological Disease

Objective and BackgroundIt is hard for some patients with pulmonary infections to get the pathogen diagnosis because of the deficiencies of traditional methods such as bacterial culture and smear staining.For metagenomic next-generation sequencing(mNGS)can obtain the genome information of pathogenic microorganisms with high throughput,high sensitivity,and short time-consuming,the role of mNGS in the pathogen diagnosis of infectious diseases has received more and more attention.Patients with malignant hematological diseases are susceptible to pulmonary infections due to immunoincompetence resulted from their primary disease,chemotherapy drugs,immunosuppressants,and other treatments.And they have a high chance of thrombocytopenia and bleeding,being more difficult to obtain lung specimens than other patients.The progress of interventional pulmonary technology has significantly improved the accuracy and safety of the biopsy of lung lesions.There are currently few comparative studies on pathogen diagnosis of pulmonary infections in patients with malignant hematological diseases between mNGS based on interventional pulmonary technology and traditional methods.In this study,mNGS based on bronchoscopic navigation(Direct Path),radial endobronchial ultrasound(radial EBUS),ultrathin bronchoscopy,and rapid on-site evaluation of cytology(ROSE)was used to diagnose pulmonary infections in patients with malignant hematological diseases,evaluate the diagnostic efficacy of mNGS and traditional methods,and further analyze the diagnostic ability of transbronchial lung biopsy(TBLB)and bronchoalveolar lavage(BAL).The aim of this study is to provide a basis for the application of more effective pathogen diagnostic methods for patients with malignant hematological diseases complicated with pulmonary infection.Methods First part,1.A retrospective and continuous collection of patients with hematological malignancies who met the inclusion criteria at the Bronchoscopy Endoscopy Center of Tianjin Medical University General Hospital from July 2018 to July 2019.Patients were clinically considering pulmonary infection.Recorded the patient’s basic diseases,clinical data,various examination results and clinical outcomes.2.The included patients were assisted by Direct Path(DP)bronchoscopic navigation,radial EBUS,ultrathin bronchoscopy,and ROSE.Obtain the patient’s lung lesion and bronchoalveolar lavage fluid(BALF)were divided into two specimens,which were submitted for traditional methods and mNGS pathogenic testing.3.Analyzed the patient’s general clinical data,initial diagnosis,traditional methods,and mNGS pathogenic test results and pathogenic distribution.Second part,1.According to the patient’s final diagnosis,compared the diagnostic efficiency of mNGS with traditional methods,including sensitivity(Se),specificity(Sp),positive predictive value(PPV),negative predictive value(NPV),and youden index.2.Analyzed the difference between the results of mNGS and traditional pathogenic detection results in patients with infectious pulmonary disease and non-infectious pulmonary disease.Third part,1.Analyzed the pathogenic difference of mNGS detection in TBLB and BAL in patients with infectious pulmonary disease and non-infectious pulmonary disease.2.Compared the diagnostic efficiency of TBLB and BAL between the two methods,including Se,Sp,PPV and NPV,and ROC curve analysis.Results First part,1.A total of 81 patients met the inclusion criteria.The patient’s initial diagnosis of pneumonia,tuberculosis,pulmonary aspergillosis,and pneumocystis jiroveci pneumonia.On chest CT examination,13.58% were patches of exudation shadows,17.28% were consolidation shadows,20.99% were diffuse ground-glass shadows,12.35% were hollow shadows,12.35% were nodules and cord shadows,8.64% were mass shadows,and 14.81% were mixed changes.2.The detection rate of traditionally cultured pathogens was 23.61%(51/216).The pathogenic bacteria were Enterococcus faecium,Pseudomonas aeruginosa,Acinetobacter baumannii,Klebsiella pneumoniae,Enterobacter cloacae,Candida albicans,Candida tropicalis,etc.3.The positive rates of pathogenic microorganisms in lung tissue and BALF mNGS sequencing were 88.89% and 97.53%,respectively.The number of detected strains were 108 and 117,respectively.The pathogenic bacteria were human cytomegalovirus,Pneumocystis jiroveci,Aspergillus and Mycobacterium tuberculosis,Enterococcus faecium,Pseudomonas aeruginosa,Acinetobacter baumannii,Klebsiella pneumonia,etc.Rare pathogenic microorganisms such as nontuberculous mycobacteria,Chlamydia psittaci,Nocardia,Rhizopus,etc.were also detected.Second part,1.The 81 patients were finally diagnosed with 11 cases of non-infectious pulmonary disease(including GVHD,IPS,and DPTS)and 70 cases of infectious pulmonary disease(including pulmonary aspergillosis,Pneumocystis jiroveci pneumonia,tuberculosis,bacterial pneumonia,cytomegalovirus pneumonia,rare pathogen infection,mixed pulmonary infection,etc.).2.The traditional pathogenic detection methods failed to effectively detect pathogenic microorganisms such as Aspergillus,Nocardia,Chlamydia psittaci,human cytomegalovirus,and some Pneumocystis jiroveci.3.The 81 patients were finally diagnosed with pulmonary aspergillosis,Pneumocystis jiroveci pneumonia,tuberculosis,bacterial pneumonia,cytomegalovirus pneumonia,rare pathogen infection,mixed pulmonary infection,GVHD,IPS,and DPTS.After traditional methods and mNGS detection,86.42%(70/81)patients received a clear pathogenic diagnosis.40.00%(28/70)were diagnosed by traditional methods,and 60.00%(42/70)were further diagnosed by mNGS.4.The traditional methods of Se and Sp were 40.00% and 63.64%,respectively,and PPV and NPV were 87.50% and 14.29%,respectively.The Se and Sp of mNGS were90.00% and 45.45%,respectively,and the PPV and NPV were 91.30% and 41.67%,respectively.The sensitivity of mNGS was better than the traditional method(P<0.05),and the specificity of mNGS was equivalent to the traditional method(P>0.05).The youden index of mNGS diagnosis is 0.355,which was better than the traditional method of 0.036.Third part,1.The Se and Sp of TBLB mNGS were 87.14% and 45.45%,respectively,and the PPV and NPV were 93.85% and 43.75%,respectively.The Se and Sp of BAL mNGS were 75.71% and 72.73%,respectively,and the PPV and NPV were 96.36% and32.00%,respectively.The sensitivity of TBLB mNGS was better than BAL(P<0.05).The specificity of BAL mNGS was better than TBLB(P<0.05).2.ROC curve analysis showed that the AUC(area under the curve)of TBLB,BAL,and TBLB-BAL combined were 0.661,0.767,and 0.782,respectively.There was no statistical difference between the three and between the two(P>0.05).Conclusion1.Pulmonary TBLB and BAL specimens can be safely obtained through interventional pulmonary technologies in patients with malignant hematological diseases and pulmonary disorders.In this study,pulmonary infections in such patients are mainly pulmonary aspergillosis,pneumocystis jiroveci pneumonia,tuberculosis,bacterial pneumonia,cytomegalovirus pneumonia,and mixed pulmonary infection.2.The sensitivity and Youden index of mNGS based on TBLB and BAL in the diagnosis of patients with malignant hematological diseases and pulmonary diseases are higher than traditional detection methods.3.The diagnostic sensitivity of mNGS from TBLB is higher than that of BAL,but the specificity of mNGS is lower than that of BAL.ROC curve analysis suggests no significant differences in the diagnostic efficacy between TBLB and BAL in patients with malignant hematological diseases and lung diseases,which needs to be further verified by prospective multi-center studies.