王成军,陈娟娟,刘洪千

二代测序技术（NGS）为肺结核诊断与耐药分析提供了突破性工具。本文综述了NGS在结核病诊疗中的应用价值及挑战。传统诊断方法如痰涂片镜检敏感性低（22%-43%），痰培养耗时长（2-8周），分子检测（如GeneXpert）虽快速但存在耐药基因覆盖不足等问题。NGS通过高通量测序实现高灵敏度（尤其低菌量样本）和特异性，可无偏倚检测结核分枝杆菌（MTB）及混合感染病原体，并同步解析耐药相关基因突变（如rpoB、katG），显著缩短耐药分析周期。全基因组测序（WGS）结合分型技术还能追踪传播链与耐药进化。然而，NGS临床应用受限于高成本、样本宿主DNA干扰、数据分析复杂性及标准化缺失。未来需整合三代测序技术优化检测效率，开发智能化分析流程，并通过技术简化与成本控制推动其在资源匮乏地区的普及。NGS的迭代与多组学联合应用有望为终结结核病提供精准化解决方案。

肺结核；二代测序技术

[1] Abel,L.,et al.,Genetics of human susceptibility to active and latent tuberculosis:present knowledge and future perspectives.Lancet Infect Dis,2018.18(3):p.e64-e75. [2] Dheda,K.,et al.,The intersecting pandemics of tuberculosis and COVID-19:population-level and patient-level impact,clinical presentation,and corrective interventions.Lancet Respir Med,2022.10(6):p.603-622. [3] Coulibaly,G.,et al.,Use of light-emitting diode fluorescence microscopy to detect acid-fast bacilli in sputum as proficient alternative tool in the diagnosis of pulmonary tuberculosis in countries with limited resource settings.Int J Mycobacteriol,2023.12(2):p.144-150. [4] Mustafa,H.,et al.,Role of Gene Xpert in smear negative pulmonary tuberculosis.Indian J Tuberc,2022.69(4):p.552-557. [5] Ajantha,P.,et al.,Urinary lipoarabinomannan in individuals with sputum-negative pulmonary tuberculosis.Indian J Med Res,2024.159(2):p.206-212. [6] Asare,K.K.,et al.,Comparison of microscopic and xpert MTB diagnoses of presumptive mycobacteria tuberculosis infection: retrospective analysis of routine diagnosis at Cape Coast Teaching Hospital.BMC Infect Dis,2024.24(1):p.660. [7] Kessel,J.,et al.,Time discrepancy for tuberculosis-negative microscopy and culture-the diagnostic gap remains:systematic analysis from a large tertiary care tuberculosis-clinic,Germany 2013-2017.Int J Infect Dis,2023.134:p.269-272. [8] Sailo,C.V.,et al.,MGIT sensitivity testing and genotyping of drug resistant Mycobacterium tuberculosis isolates from Mizoram, Northeast India.Indian J Med Microbiol,2022.40(3):p.347-353. [9] Shah,B.,et al.,The clinician,the lab and the patient:Understanding lab diagnostics to eradicate tuberculosis.Indian J Tuberc,2023.70(1):p.42-48. [10] Yao,L.,et al.,The diagnostic performance of endobronchial ultrasound with Xpert MTB/RIF Ultra in smear-negative pulmonary tuberculosis.BMC Infect Dis,2023.23(1):p.107. [11] Li,Z.,et al.,The incremental value of Mycobacterium tuberculosis trace nucleic acid detection in CT-guided percutaneous biopsy needle rinse solutions for the diagnosis of tuberculosis.Front Microbiol,2024.15:p.1335526. [12] Nathavitharana,R.R.,et al.,Assessing Infectiousness and the Impact of Effective Treatment to Guide Isolation Recommendations for People With Pulmonary Tuberculosis.J Infect Dis,2025.231(1):p.10-22. [13] Gao,M.,et al.,Advancements in LAM-based diagnostic kit for tuberculosis detection:enhancing TB diagnosis in HIV-negative individuals.Front Microbiol,2024.15:p.1367092. [14] Huang,H.C.,et al.,Novel TB smear microscopy automation system in detecting acid-fast bacilli for tuberculosis-A multi-center double blind study.Tuberculosis(Edinb),2022.135:p.102212. [15] Omrani,A.S.,et al.,GeneXpert MTB/RIF Testing in the Management of Patients with Active Tuberculosis;A Real Life Experience from Saudi Arabia.Infect Chemother,2014.46(1):p.30-4. [16] Nikam,C.,et al.,Evaluation of the Indian TrueNAT micro RT-PCR device with GeneXpert for case detection of pulmonary tuberculosis. Int J Mycobacteriol,2014.3(3):p.205-10. [17] Mokaddas,E.M.,H.Saadaldeen,and S.Ahmad,Comparison of two molecular methods and an automated liquid culture system for the early detection of Mycobacterium tuberculosis from both pulmonary and extrapulmonary specimens in Kuwait.Int J Mycobacteriol,2016.5 Suppl 1:p.S74-s75. [18] Dahiya,B.,et al.,Diagnosis of extrapulmonary tuberculosis by GeneXpert MTB/RIF Ultra assay.Expert Rev Mol Diagn,2023.23(7):p.561-582. [19] Marouane,C.,et al.,Evaluation of molecular detection of extrapulmonary tuberculosis and resistance to rifampicin with GeneXpert® MTB/RIF.Med Mal Infect,2016.46(1):p.20-4. [20] Hashmi,A.A.,et al.,Utility of the GeneXpert Mycobacterium tuberculosis/Rifampin(MTB/RIF)Assay on Paraffin-Embedded Biopsy Tissue Samples for Detecting Tuberculosis:Comparison With Histopathology.Cureus,2020.12(12):p.e12048. [21] Sharma,K.,et al.,Detection of viable Mycobacterium tuberculosis in ocular fluids using mRNA-based multiplex polymerase chain reaction.Indian J Med Microbiol,2022.40(2):p.254-257. [22] Sah,A.K.,et al.,Comparative Study of GeneXpert MTB/RIF Assay and Multiplex PCR Assay for Direct Detection of Mycobacterium tuberculosis in Suspected Pulmonary Tuberculosis Patients.Curr Microbiol,2017.74(9):p.1026-1032. [23] Kamra,E.,et al.,Diagnosis of urogenital tuberculosis by multiplex-nested PCR targeting mpt64(Rv1980c)and IS6110:comparison with multiplex PCR and GeneXpert®MTB/RIF.Lett Appl Microbiol,2022.75(4):p.857-868. [24] Bodmer,T.and A.Ströhle,Diagnosing pulmonary tuberculosis with the Xpert MTB/RIF test.J Vis Exp,2012(62):p.e3547. [25] Li,J.,et al.,Establishment and evaluation of an overlap extension polymerase chain reaction technique for rapid and efficient detection of drug-resistance in Mycobacterium tuberculosis.Infect Dis Poverty,2022.11(1):p.31. [26] Üzer,F.,et al.,The clinical utility of tuberculin skin tests:a single-center experience.Colomb Med(Cali),2022.53(3):p.e2015055. [27] Seyhoglu,E.,et al.,QuantiFERON®-TB Gold In-Tube test can be used for screening latent tuberculosis before biological treatment in a Bacille Calmette-Guérin(BCG)-vaccinated country:the HUR-BIO single-center real-life results.Clin Rheumatol,2021.40(5):p.2027-2035. [28] Singh,R.,et al.,Evaluation of role of interferon gamma release assays in the diagnosis of latent tuberculosis in human immunode- ficiency virus-infected patients.Indian J Sex Transm Dis AIDS,2021.42(2):p.111-117. [29] Benachinmardi,K.,S.Sampath,and M.Rao,Evaluation of a new interferon gamma release assay,in comparison to tuberculin skin tests and quantiferon tuberculosis goldplus for the detection of latent tuberculosis infection in children from a high tuberculosis burden setting. Int J Mycobacteriol,2021.10(2):p.142-148. [30] Petnak,T.,et al.,Diagnostic accuracy of interferon-gamma release assays for diagnosis of smear-negative pulmonary tuberculosis:a systematic review and meta-analysis.BMC Pulm Med,2022.22(1):p.219. [31] Zhang,Y.,et al.,Comparing the diagnostic performance of QuantiFERON-TB Gold Plus with QFT-GIT,T-SPOT.TB and TST:a systematic review and meta-analysis.BMC Infect Dis,2023.23(1):p.40. [32] Rong,Y.,et al.,Diagnostic value of interferon-γrelease assay in patients with COPD complicated with pulmonary tuberculosis.BMC Infect Dis,2025.25(1):p.99. [33] Nolan,D.J.,et al.,Comparing Gold-Standard Sanger Sequencing with Two Next-Generation Sequencing Platforms of HIV-1 gp160 Single Genome Amplicons.AIDS Res Hum Retroviruses,2024.40(11):p.659-669. [34] McDonald,A.L.,et al.,Efficient small fragment sequencing of human,cattle,and bison miRNA,small RNA,or csRNA-seq libraries using AVITI.BMC Genomics,2024.25(1):p.1157. [35] Bestard-Cuche,N.,et al.,Illumina SBS Sequencing and DNBSEQ Perform Similarly for Single-Cell Transcriptomics.Genes(Basel),2024.15(11). [36] Goraichuk,I.V.,et al.,Improved influenza A whole-genome sequencing protocol.Front Cell Infect Microbiol,2024.14:p.1497278. [37] Schwab,T.C.,et al.,Targeted next-generation sequencing to diagnose drug-resistant tuberculosis:a systematic review and meta-analysis. Lancet Infect Dis,2024.24(10):p.1162-1176. [38] El-Lagta,N.,et al.,Revolutionising High Resolution HLA Genotyping for Transplant Assessment:Validation,Implementation and Challenges of Oxford Nanopore Technologies'Q20(+)Sequencing.Hla,2024.104(4):p.e15725. [39] Golparian,D.,et al.,Novel approach using automated target enrichment enables culture-independent accurate whole-genome sequencing of Neisseria gonorrhoeae directly from clinical urogenital and extragenital specimens.J Antimicrob Chemother,2025.80(2):p.563-566. [40] Davidson,I.M.,et al.,Methodological approaches in 16S sequencing of female reproductive tract in fertility patients:a review.J Assist Reprod Genet,2025.42(1):p.15-37. [41] Haarkötter,C.,et al.,Exploring the Potential of Genome-Wide Hybridization Capture Enrichment for Forensic DNA Profiling of Degraded Bones.Genes(Basel),2024.16(1). [42] Chaudhary,P.,et al.,Validation of a novel NGS based BCR::ABL1 kinase domain mutation detection assay in Indian cohort.Sci Rep,2024.14(1):p.15745. [43] Xiao,H.,et al.,Metagenomic next-generation sequencing of bronchoalveolar lavage fluid samples offers diagnostic utility in bacteriologically negative pulmonary tuberculosis.Diagn Microbiol Infect Dis,2025.111(4):p.116725. [44] Gao,W.,et al.,Nanopore-based targeted next-generation sequencing of tissue samples for tuberculosis diagnosis.Front Microbiol, 2024.15:p.1403619. [45] Wu,X.,et al.,Targeted next-generation sequencing-a promising approach in the diagnosis of Mycobacterium tuberculosis and drug resistance.Infection,2024. [46] Yuan,J.,et al.,Host DNA depletion assisted metagenomic sequencing of bronchoalveolar lavage fluids for diagnosis of pulmonary tuberculosis.Ann Clin Microbiol Antimicrob,2025.24(1):p.13. [47] Léveillé,N.,et al.,Mycobacterium tuberculosis pseudo-outbreak due to laboratory cross-contamination:A molecular epidemiology outbreak investigation.Can Commun Dis Rep,2024.50(12):p.430-435. [48] Chen,W.,et al.,A case report of confirmed difficult pulmonary tuberculosis based on the hybrid capture-based tNGS method.BMC Pulm Med,2025.25(1):p.64. [49] Tao,Y.,et al.,Diagnostic Value of Targeted Next-generation Sequencing in Pulmonary Mycobacterial Infections.Curr Med Sci, 2024.44(5):p.947-953. [50] You,Y.,Y.M.Ni,and G.Shi,Diagnostic accuracy of metagenomic next-generation sequencing in pulmonary tuberculosis:a systematic review and meta-analysis.Syst Rev,2024.13(1):p.317.

点击下载PDF