福建省肺非结核分枝杆菌临床分离株的菌种鉴定（英文）

目的对2005-2011年间,福建省福州市胸科医院分离获得的非结核分枝杆菌临床菌株进行菌种鉴定。方法分别使用传统鉴别培养基法和多位点PCR、hsp65-PRA与rpoB-PRA,以及hsp65、rpoB、16srRNA和ITS基因测序等分子生物学方法,对全部菌株进行菌种鉴定。结果 450株被初步鉴定为非结核分枝杆菌的临床分离菌株中,有45株被鉴定为结核分枝杆菌,其余405株被鉴定为23种不同的非结核分枝杆菌和1株支气管戈登菌。结论在该地区有多种非结核分枝杆菌传播和流行,其中,6种非结核分枝杆菌和支气管戈登菌为在福建省内首次发现。     

大理地区13株疑似非结核分枝杆菌临床分离株的鉴定

目的对云南大理地区13株疑似非结核分枝杆菌(Non-Tuberculous mycobacteria,NTM)进行鉴定,了解其菌种构成。方法大理州疾控中心提供的疑似非结核分枝杆菌13株,经罗氏培养基(L-J)、噻吩-2-羧酸肼(TCH)和对硝基苯甲酸(PNB)鉴别培养基初步鉴别培养确定为NTM后分别通过16S rRNA、hsp65和rpoB基因片段测序进行菌种鉴定。结果 13株疑似NTM临床分离株经16S rRNA、rpoB和hsp65基因DNA测序分析鉴定为NTM 11株(84.62%),束村氏菌1株(7.69%),戈登氏菌1株(7.69%)。11株NTM中鸟分枝杆菌(M.avium)9株,脓肿分支杆菌(M.abcessus)2株。结论经抗酸染色筛查、L-J和PNB/TCH培养基初步鉴定为NTM的临床分离株通过3种不同基因测序分析鉴定为NTM、束村氏菌和戈登氏菌,以NTM中的鸟分枝杆菌占多数。     

福建省临床分离非结核分枝杆菌菌种分布研究

目的 分析我院临床分离非结核分枝杆菌(NTM)菌株,探讨福建省NTM临床分离率和菌种分布情况。方法 对我院2009-2012年临床分离6 362株分枝杆菌进行结核分枝杆菌复合群(MTBC)与NTM鉴别,对鉴定为NTM菌株应用hsp65-和rpoB- PCR-RFLP进行菌种鉴定。结果 6 362株临床分离分枝杆菌共鉴别出649株NTM。鉴别出的649株NTM菌株,经hsp65-和rpoB-PCR-RFLP鉴定,菌种分布达24个种,其中菌株数量前3位菌种为:胞内分枝杆菌(M.intracellulare)302株(46.5%),鸟分枝杆菌(M. avium)138株(21.3%),脓肿分枝杆菌(M. abscessus)81株(12.5%)。结论 2009-2012 年福建省NTM临床分离率占分枝杆菌培养阳性菌株的10.2%,NTM种类多,以鸟-胞内分枝杆菌复合群(MAC)为福建省NTM的主要流行菌种,占总数的67.8%。     

四川省46株疑似非结核分枝杆菌临床分离株菌种鉴定结果分析

目的对四川省46株疑似非结核分枝杆菌进行菌种鉴定。方法针对四川省46份临床分离疑似非结核分枝杆菌(Nontuberculous mycobacteria,NTM) 16S rRNA基因和16S-23S rRNA内转录间隔区(ITs)进行基因测序和序列分析。结果 46株菌株中,35株鉴定为NTM (经基因测序,分别为11种菌),10株为戈登氏菌,1株为诺卡氏菌。结论四川省NTM至少有11种,以胞内分枝杆菌、脓肿分枝杆菌和堪萨斯分枝杆菌为主。NTM菌种鉴定对临床鉴别诊断和合理治疗有一定的意义。     

山东省非结核分枝杆菌感染的菌种分布研究

目的研究山东省13个哨点县2004—2007年非结核分枝杆菌临床分离率和菌种分布情况。方法对山东省13个哨点县2004—2007年送到汉光中心的2625株分枝杆菌菌株,进行分枝杆菌菌群鉴定试验,鉴定为非结核分枝杆菌的菌株应用16SrDNA测序方法进行菌种鉴定。结果2625株菌株分枝杆菌菌群鉴定39株为非结核分枝杆菌菌群,39株菌株16SrDNA序列分析结果有36株是非结核分枝杆菌,其中29株为胞内分枝杆菌株（80.6%）, 其余分别为堪萨斯分枝杆菌、偶然分枝杆菌各2株、戈登分枝杆菌、龟脓肿分枝杆菌复合物、瘰疬分枝杆菌各1株;结核分枝杆菌复合群2株;鼻疽诺卡氏菌1株。山东地区非结核分枝杆菌临床分离率占分枝杆菌培养阳性菌株的1.4%。结论山东地区流行的非结核分枝杆菌以慢生长分枝杆菌的胞内分枝杆菌为主。     

应用气相色谱技术分析全细胞脂肪酸快速鉴定分枝杆菌

目的探讨应用气相色谱技术分析全细胞脂肪酸进行分枝杆菌菌种鉴定方法的准确性和实用性。方法应用美国MIDI公司开发的基于细胞脂肪酸成分鉴定细菌菌种的软件系统MIS4．0，对14株分枝杆菌参考菌株和727株分枝杆菌临床分离株进行菌种鉴定，同时采用传统方法对相同菌株进行菌种鉴定，比较两种方法的检测结果。结果(1)参考菌株鉴定：应用传统方法对所有菌株均获得正确鉴定结果，应用脂肪酸分析方法则除母牛分枝杆菌外，其他13株均获得正确鉴定结果。(2)临床分离株鉴定：传统方法鉴定为结核分枝杆菌、牛分枝杆菌的625株中，应用脂肪酸分析方法有45株鉴定为非结核分枝杆菌，对结核分枝杆菌鉴定的正确率为93％；对传统方法鉴定为非结核分枝杆菌的102株菌株，脂肪酸分析法亦全部鉴定为非结核分枝杆菌，但其中7株的菌种鉴定结果不同，其鉴定正确率为93％。(3)脂肪酸分析法对结核分枝杆菌鉴定错误者主要是将其鉴定为胃分枝杆菌、次要分枝杆菌和耻垢分枝杆菌，对非结核分枝杆菌则主要是将瘰疬分枝杆菌鉴定为戈登分枝杆菌。结论应用气相色谱技术分析全细胞脂肪酸进行分枝杆菌菌种鉴定，和传统鉴定方法结果具有良好的一致性，且可通过一次性实验操作将临床中常见的分枝杆菌鉴定到种，是一种准确度高、实用性强的分枝杆菌菌种鉴定方法。     

43株甘肃临床分离非结核分枝杆菌分类鉴定

目的了解甘肃当前临床流行的非结核分枝杆菌(NTM)的病原谱特点及其主要NTM种类,为指导临床诊疗、有效防治NTM肺病提供参考依据。方法收集2012年~2014年来源于甘肃省不同地区的875株临床分离分枝杆菌菌株,经PNB/TCH方法初步鉴定为疑似NTM菌株,采用16SrRNA基因测序技术进行菌种鉴定。结果 875株分枝杆菌临床分离菌株中分离出疑似NTM菌株46株。经16SrRNA基因序列分析鉴定,43株为NTM,3株为诺卡氏菌。43株NTM菌株分别为胞内分枝杆菌、堪萨斯分枝杆菌、塞内加尔分枝杆菌、鸟分枝杆菌、戈登氏分枝杆菌、楚尔盖分枝杆菌、罕见分枝杆菌、偶然分枝杆菌和脓肿分枝杆菌。其中,胞内分枝杆菌有31株,占总NTM菌株数的72.09%。结论甘肃省非结核分枝杆菌群以胞内分枝杆菌为主,应用16SrRNA基因序列分析鉴定方法能准确鉴定出NTM菌种,为临床诊治提供依据。     

某高级中学结核病爆发分离菌株的实验室鉴定

目的鉴定从某中学结核病爆发分离菌株的种属。方法对2006年5—8月,辽宁省某高级中学发生的10例痰检阳性病例中的3份痰标本分离的菌株经表型鉴定方法;传统生化方法;扩增hupB基因、dnaA-dnaN 和NTF-1区;Spoligotyping 以及MIRU基因分型;16S rRNA基因、16S-23S ITS和hsp65基因测序以及hsp65基因限制性酶切分析进行鉴定。结果临床分离株经生化方法初步鉴定1份为结核分枝杆菌复合群和2份为非结核分枝杆菌,随后经表型鉴定和扩增hupB基因、dnaA-dnaN 和NTF-1区;Spoligotyping 以及MIRU基因分型,说明属于结核分枝杆菌复合群的菌株为结核分枝杆菌北京基因型现代株,MIRU基因型为223325173533。经16S rRNA基因、16S-23S ITS和hsp65基因测序以及hsp65基因限制性酶切分析说明属于非结核分枝杆菌的菌株为猪分枝杆菌。结论本次从结核病爆发累及病人的标本中分离出结核分枝杆菌北京基因型现代株和猪分枝杆菌,猪分枝杆菌在暴发流行中的意义尚需进一步研究。     

hsp65 and rpoB PCR-RFLP用于龟/脓肿分枝杆菌复合群种的快速鉴定(英文)

目的探讨和评价hsp65and rpoB PCR-RFLP用于龟/脓肿分枝杆菌复合群种的快速鉴定。方法收集经PNB/TCH鉴别培养基表型鉴定和16s rRNA基因测序鉴定为龟/脓肿分枝杆菌复合群的临床分离菌株,用hsp65and rpoBPCR-RFLP进行种/亚种鉴定。结果经表型鉴定为非结核分枝杆菌的27株临床菌株,16s rRNA基因测序分析与龟/脓肿分枝杆菌的同源性达到99.7%。经hsp65PCR-RFLP and rpoBPCR-RFLP鉴定18株为脓肿分枝杆菌(M.abscessus),4株为溃疡分枝杆菌(M.absecces),另5株表现为独特的指纹特征,可能是一个新的亚种。结论能够快速进行龟/脓肿分枝杆菌复合群种/亚种的鉴定。     

多位点PCR用于安徽391株分枝杆菌分离株的快速鉴定

摘要：目的 评价多位点PCR用于分枝杆菌临床分离株快速鉴定效果。方法 收集从临床结核病患者分离到的抗酸染色阳性的培养物,经PNB/TCH鉴别培养基进行培养鉴定后,采用聚合酶链反应(PCR)对16SrRNA、Rv0577、IS1561、Rv1510、Rv1970、Rv3877/8和Rv3120 基因位点进行扩增,鉴定至种,再经rpoB-PRA、hsp65和rpoB基因测序进行验证。结果 共对391株分枝杆菌临床分离株应用多位点PCR进行了鉴定,结果显示结核分枝杆菌378株,非洲分枝杆菌I型6株,非结核分枝杆菌7株。7株非结核分枝杆菌分别为鸟分枝杆菌1株,马赛分枝杆菌2株,胞内分枝杆菌4株。而PNB/TCH鉴别培养基培养鉴定结果为结核分枝杆菌复合群385株,非结核分枝杆菌6株。多位点PCR结果与rpoB-PRA、hsp65和rpoB基因测序结果一致。结论 多位点PCR技术鉴定分枝杆菌菌种结果准确可靠,且具有简便和快速等优点,有较大的分子流行病学应用价值,且对于临床诊断和治疗都具有重要意义。     

Identification and characterization of nontuberculous mycobacteria isolated from suspected pulmonary tuberculosis patients in eastern china from 2009 to 2019 using an identification array system

ObjectiveNontuberculous mycobacteria (NTM) species are increasingly being isolated and have become a key factor affecting public health by causing pulmonary diseases. Most NTM species do not respond to conventional tuberculosis (TB) drugs. This study aimed to identify NTM isolated from suspected pulmonary TB patients from the Zhejiang province and analyze their distribution in the region.MethodsA total of 1,113 NTM isolates from patients suspected to be suffering from acid-fast bacilli-positive tuberculosis were identified at the species level, using the CapitalBio Mycobacterium identification array and polymerase chain reaction amplification and sequencing of 16S-23S gene internal transcribed spacer (ITS), 16S rRNA, and hsp65.ResultsOf the 23,138 isolates, we identified 1,102 NTM (4.8%), mainly including Mycobacterium intracellulare (54.81%, 604/1,102), M. chelonae-M. abscessus (16.52%, 182/1,102), M. avium (13.16%, 145/1,102), M. kansasii (8.17%, 90/1,102), and M. gordonae (3.27%, 36/1,102).ConclusionThe distribution of NTM species observed in patients with suspected pulmonary tuberculosis provides guidance for the diagnosis and treatment of NTM pulmonary diseases.     

Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from non-tuberculous mycobacteria

Most mycobacterial infections are still caused by Mycobacterium tuberculosis complex (MTC) strains; however, infections by non-tuberculous mycobacteria (NTM) are increasing, particularly among immunocompromised patients. Conventional species-specific identification and proper patient management are delayed due to the slow-growing nature of mycobacteria. We have developed a multiplex PCR (mPCR) targeting the oxyR-ahpC intergenic region and rpoB gene for direct detection and differentiation of clinical isolates as MTC or NTM in primary culture. Two amplicons of 473 bp and 235 bp from MTC members and a single amplicon of 136 bp from NTM are expected. The mPCR was developed using several mycobacterial species and was evaluated by testing extracted DNA from liquid cultures, flagged as positive for bacterial growth, of 100 consecutive mycobacterial isolates. The results were validated by DNA sequencing of the species-specific 16S-23S internal transcribed spacer (ITS) region. The mPCR with template DNA from reference Mycobacterium spp. yielded the expected amplicons. When 100 consecutive clinical isolates of Mycobacterium spp. were tested, 92 strains yielded MTC member-specific amplicons, and DNA sequences from 10 randomly selected isolates matched completely with the ITS sequence from M. tuberculosis. Eight isolates were identified as NTM, and DNA sequencing of the ITS region confirmed the NTM status of each of these isolates. The mPCR developed in this study allowed rapid detection and differentiation of primary cultures as MTC or NTM, thus helping in timely institution of specific therapy.     

西安市非结核分枝杆菌感染现状调查分析

目的 了解西安市结核病患者中非结核分枝杆菌感染情况。方法 收集西安市胸科医院检验科分离培养的分枝杆菌临床菌株,利用两步PCR方法及PCR产物直接测序的方法进行菌种鉴定。结果 经鉴定发现,收集的202株分枝杆菌中有195株为结核分枝杆菌,3株为牛结核分枝杆菌,2份为鸟分枝杆菌,1份为堪萨斯分枝杆菌,1份为灰色链霉菌,非结核分枝杆菌感染率为1.5%(3/201)。结论 西安市尚存在一定比例非结核分枝杆菌感染,进一步菌种鉴定有利于临床正确诊断和合理用药治疗。     

Evaluation of 120 mycobacterial strains isolated from clinical specimens to the species level by polymerase chain reaction-restriction enzyme analysis

In this study, a total of 120 mycobacterial strains isolated from clinical specimens in Hacettepe University Hospital Clinical Pathology Laboratories were evaluated by polymerase chain reaction-restriction enzyme analysis (PRA), which analyses the common mycobacterial heat shock protein gene (hsp65). 95 of 120 strains (79.1%) were identified as Mycobacterium tuberculosis and 25 (20.8%) were identified as non-tuberculous mycobacteria (NTM). M. gordonae I and IV were the most common NTM species (3.3% each) followed by M. chelonae (2.5%). Other NTM species isolated were M. gordonae III, M. avium, M. peregrinum (1.6%), M. fortuitum, M. flavescens, M. malmoense and M. mucogenicum (0.8%). Four isolates had PRA patterns that did not match any patterns previously described. The patients who had NTM had underlying diseases; the most frequent clinical diagnosis among these was chronic obstructive pulmonary disease (COPD) and chronic renal failure. AIDS and pulmonary carcinoma were the other underlying diseases detected.     

A rapid loop-mediated isothermal amplification assay targeting hspX for the detection of Mycobacterium tuberculosis complex

A rapid, simple, and low-cost diagnostic tool for tuberculosis (TB) detection is urgently needed in countries with a high TB burden. Here, we report a novel loop-mediated isothermal amplification (LAMP) assay targeting the hspX gene for the rapid detection of Mycobacterium tuberculosis, M. bovis, M. africanum, and M. microti. The specificity of this assay was evaluated using 4 reference strains of Mycobacterium tuberculosis complex (MTC), 22 species of non-tuberculous mycobacteria (NTM), 7 non-mycobacterial species, and 50 clinical M. tuberculosis isolates. All the reference MTC strains and M. tuberculosis clinical isolates were successfully detected by this method, and there were no false-positive results with NTM or non-mycobacterial species, which demonstrates the high specificity of this assay for MTC. The detection limit was 10 copies of MTC genome within 27 min, and the detection speed of this assay was higher than that of any other isothermal methods reported so far. Because of its speed, simplicity, sensitivity, specificity, and inexpensiveness, the TB hspX LAMP assay is a potential gene diagnostic method for TB detection in developing countries with a high TB burden.     

湖南省525株非结核分枝杆菌临床分离株的菌种鉴定与流行特征

目的 对2019-2020年湖南省分离的非结核分枝杆菌(NTM)进行菌种鉴定,并分析其分布特征,为非结核分枝杆菌病的防治提供基础科学依据。方法 收集2019-2020年湖南省胸科医院疑似结核病患者分离到的分枝杆菌临床分离株,通过MPB64蛋白免疫胶体金法、PNB(对硝基苯甲酸)培养基生长试验进行结核分枝杆菌复合群和NTM鉴定,应用16S rRNA和Hsp65测序分析法对NTM菌株进行菌种鉴定。结果 共收集到6 515份分枝杆菌阳性培养物,初步鉴定为NTM共525株,占比8.06%,其中分离于男性患者285株,占比54.29%;女性患者240株,占比45.71%;人群分布以农民为主,共367株,占比69.90%;年龄分布以40岁以上中老年患者最多,共402株,占比76.57%。2019-2020年NTM菌种分布达27种,菌种分布位列前4者分别为脓肿分枝杆菌(190株,36.19%)、胞内分枝杆菌(174株,33.14%)、鸟分枝杆菌(64株,12.19%)、戈登分枝杆菌(47株,8.95%);其它50株(9.52%)包含23种NTM。脓肿分枝杆菌主要在郴州市、湘西自治州地区、衡阳市和永州市等地区流行;胞内分枝杆菌主要在郴州市、岳阳市和湘潭市流行。结论 2019-2020年湖南地区分枝杆菌中NTM检出率仍保持较高水平,以脓肿分枝杆菌、胞内分枝杆菌、鸟分枝杆菌和戈登分枝杆菌为主;感染人群以男性、中老年、农民为主。