噬菌体法检测结核分枝杆菌乙胺丁醇耐药性的研究

目的 建立噬菌体生物扩增法（PhaB法）快速测定乙胺丁醇耐药性的实验体系,探讨PhaB法在测定结核分枝杆菌乙胺丁醇耐药性的应用价值.方法 应用PhaB法测定115株临床分离结核分枝杆菌耐药性,并与BacT/ALERT 3D药敏试验结果相比较,对耐药性测定结果不一致的菌株测定其最低抑菌浓度.结果 115株结核分枝杆菌临床分离株中经BacT/ALERT 3D测定敏感89株,耐药26株;PhaB法测定敏感93株,耐药22株.两种方法测定均为敏感87株,均为耐药20株,两种方法测定药敏结果相符率为93.0%;结果不符8株,不符合率6.96%.如以BacT/ALERT 3D药敏结果为判断标准,则PhaB法检测乙胺丁醇耐药性的敏感性为76.9%（20/26）,特异性为97.8%（87/89）,阳性预测值为90.9%（20/22）,阴性预测值为93.5%（87/93）,准确性为93.0%（107/115）.结论 PhaB法检测结核分枝杆菌乙胺丁醇耐药性只需3天时间,操作简便,不需特殊仪器设备,可作为结核分枝杆菌乙胺丁醇耐药性快速筛选方法之一.     

MPB64抗原检测快速诊断结核分枝杆菌的临床价值

目的比较痰涂片抗酸染色、改良酸性罗氏培养、MPB64的免疫胶体金法用于检测结核分枝杆菌特异性的分泌蛋白质MPB64,评价MPB64的免疫胶体金法在结核分枝杆菌快速检测中的应用价值。方法选取438例确诊肺结核患者痰液,进行痰涂片抗酸染色、7H9液体培养基和改良酸性罗氏培养基培养、基于MPB64的免疫胶体金法检测,对结果进行比较分析。并选取痰涂片确认为阳性的标本33例,用米氏7H9液体培养和基于MPB64的免疫胶体金法进行检出时间对比。结果痰涂片阳性率为14.8%,罗氏培养阳性率为20.7%,7H9液体培养阳性率24.8%,MPB64的免疫胶体金法阳性率为25.1%。基于MPB64的免疫胶体金法在培养的第15天检出32例阳性;而单纯的7H9液体培养加抗酸染色检测法在第15天只检出11例阳性。结论7H9液体培养配合基于MPB64的免疫胶体金法可以作为一种较为敏感和特异的检测结核分枝杆菌感染的方法,并可以有效的区分结核分枝杆菌和非结核分枝杆菌。     

PCR法鉴定结核分枝杆菌复合群亚种的研究

结核分枝杆菌复合群中各菌型致病性和药物敏感性不尽相同,在临床治疗前需要进行菌种鉴定,目前临床使用的生化方法耗时长,给治疗带来不便,本研究采用PCR法可快速鉴定结核分枝杆菌复合群亚种,具有快速、准确的优点,现将研究结果报道如下。1材料和方法1.1材料1.1.1菌株人结核分枝杆菌标准株(H37Rv,CMCC(B)95053)和牛结核分枝杆菌标准株(CMCC(B)93006),非洲分枝杆菌标准株(M.africanum,CMCC(B)95049)购自国家菌种保存中心。19株牛结核分枝杆菌分离株(M.bovis),PNB培养生长试验阴性,TCH培养生长试验为阴性;20株人结核分枝杆菌分离株(M.tb) 更多还原     

云南省AIDS合并TB病原学诊断与耐药性

目的提高艾滋病（AIDS）合并结核病（TB）的病原学诊断的阳性率,获得AIDS合并TB临床分离结核分枝杆菌菌株对一线抗结核药物的耐药率。方法采用萋-尼抗酸染色、BacT/ALERT3D360MP结核分枝杆菌培养和实时荧光定量聚合酶链反应（PCR）,对AIDS合并疑似结核分枝杆菌感染病人不同时段的样本同时进行检测;对结核分枝杆菌培养阳性物,经抗酸染色和TB-DNAPCR确认为结核分枝杆菌的进行抗结核药敏试验。结果涂片萋-尼抗酸染色阳性率为11.69%（18/154）;样本直接进行TB-DNAPCR扩增的阳性率18.18%（28/154）;MP培养阳性经抗酸染色和TB-DNAPCR确认结核分枝杆菌的阳性率20.13%（31/154）。临床分离株对抗结核一线药物的敏感试验结果显示,异烟肼的耐药率高达81.48%,其次是链霉素37.04%,乙胺丁醇和利福平分别为25.93%、18.52%。结论云南省AIDS合并结核分枝杆菌临床分离率为20.13%（31/154）,AIDS合并非结核分枝杆菌的临床分离率为16.23%（25/154）。实时荧光PCR扩增TB-DNA能为临床提供早期病原学诊断依据,而结核分枝杆菌培养在提供病原学诊断＂金标准＂的同时,可为进一步检测其耐药性准备菌株。鉴于AIDS病人免疫缺陷的特殊性,需要采用多种方法对多份样本和多种类样本同时检测,提高检测的特异性和敏感性。     

分泌蛋白MPT64鉴定结核分枝杆菌复合群的应用研究

v、牛分枝杆菌和非洲分枝杆菌为阳性,24株非MTB均为阴性;mpt64基因扩增的结果与ELISA法检测结果完全相符.ELISA法检测MTB临床分离株的敏感度为97.3%(110/113),57株非MTB均未检出MPT64蛋白,检测特异度为100.0%.基因扩增MTB中2株为阴性,非MTB均为阴性.结论 采用ELISA法检测分泌蛋白MPT64来鉴定MTB复合群,是一种准确、快速和简便的方法,值得临床推广应用.     

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。     

快速结核分枝杆菌菌型鉴定方法的应用评估

目的评估MGIT 960液体培养分枝杆菌阳性时,免疫胶体金法MPB64抗原检测及荧光探针PCR法在结核分枝杆菌快速菌型鉴定中的应用价值。方法临床MGIT960TM液体培养得到203例分枝杆菌阳性菌株,平行采用上述两种方法进行结核分枝杆菌菌型鉴定。结果免疫胶体金MPB64抗原检测法灵敏度为97.7%(169/173×100%),特异度为100%(30/30×100%);荧光探针PCR法灵敏度为100%(173/173×100%),特异度为96.7%(29/30×100%)。结论免疫胶体金法检测MPB64抗原及荧光探针PCR法作为液体培养时结核杆菌菌型鉴定的方法均是快速可靠的。免疫胶体金法检测MPB64抗原由于操作简便,实验要求条件低,更易为临床TB实验室接受。     

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。     

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。     

上海市某结核病定点医院结核病房空气样本中分枝杆菌的检测

目的 了解上海市某结核病定点医院结核病房空气中的分枝杆菌污染情况及潜在传染性。 方法 采用液体撞击式微生物气溶胶采样器（FA-1型撞击式空气微生物采样器）采集该医院22间结核病房中空气样本134份,均在痰涂片抗酸杆菌阳性（＋~＋＋＋＋）患者床边采集。每间病房均有4张床位,每间病房的空间布局相同,但患者组成情况不完全相同。经氢氧化钠-N-乙酰-L-半胱氨酸(NALC-NaOH)前处理,分别采用罗氏培养、BacT/ALERT 3D全自动快速培养进行分枝杆菌分离培养,并对培养阳性样本采用DNA序列分析的方法进行菌型鉴定。两种分离培养方法对医院空气样本中分枝杆菌分离率的比较采用配对卡方检验,以P<0.05为差异有统计学意义。 结果 134份结核病房涂阳患者床边采集到的空气样本中有3份分枝杆菌分离培养阳性,经DNA序列分析证实分别为结核分枝杆菌、胞内分枝杆菌和戈登分枝杆菌。BacT/ALERT 3D培养阳性3份,经DNA序列分析证实分别为结核分枝杆菌、胞内分枝杆菌和戈登分枝杆菌,污染2份,阳性检出率为2.3%（3/132）。罗氏培养阳性2份,经DNA序列分析证实分别为胞内分枝杆菌和戈登分枝杆菌,污染1份,阳性检出率为1.5%(2/133)。经配对卡方检验,两种分离培养方法对医院空气样本中分枝杆菌分离率差异无统计学意义（χ²=0.00,P>0.05）。 结论 该医院结核病房痰涂片抗酸杆菌阳性患者床边所采集的空气样本中可分离到分枝杆菌,虽然目前尚无法判断这些菌株是否由于就诊患者传播所致,但是定期监测医院内空气质量是预防与控制医院内感染的一项重要措施。     

BACTEC MGIT 960和BACT/ALERT 3D系统快速培养和鉴定痰中抗酸杆菌

目的对抗酸染色阳性痰行分枝杆菌培养和鉴定。方法采用萋一尼氏抗酸染色法对临床诊断肺结核患者的晨痰涂片直接镜检，抗酸染色阳性痰经BACTEC960和BACT/ALERT3D系统进行分枝杆菌培养，分别经对硝基苯甲酸（PNB）和噻吩-2-羧基肼（TCH）培养基生长试验行分枝杆菌菌群和结核分枝杆菌复合群菌种鉴定。结果抗酸染色阳性痰标本的分枝杆菌培养阳性率100％，大多数为结核分枝杆菌（9／10），少数为非结核分枝杆菌（1／10），最快6天即可报告分枝杆菌阳性培养。结论BACTEC 960和BACT／ALERT3D系统具有快速培养分枝杆菌作用，抗酸染色阳性痰有必要行分枝杆菌培养和鉴定，有利于肺结核与非结核分枝杆菌病的鉴别诊断、结核分枝杆菌菌种鉴定和抗结核药物敏感性试验。     

两种结核分枝杆菌培养检测方法的比较

目的比较小川培养基和BacT/ALERT3D系统培养检测结核分枝杆菌结果的差异。方法对100份结核患者痰标本进行前处理后,分别用上述两种方法进行培养。结果两种方法的阳性率分别为28%、38%;阳性平均报告时间分别为28.3天、14.6天;污染率分别为3%、5%。结论3D系统培养法较小川培养法有更高的灵敏度和更快的检出时间,在加强无菌操作的情况下,3D系统可以取代小川培养法,作为结核杆菌培养检测的常规方法。     

2004～2009年我中心结核分枝杆菌临床株耐药趋势

目的探讨重庆市公共卫生医疗救治中心临床结核菌株耐药趋势。方法采用BACT/ALERT3D进行各种临床标本的分枝杆菌分离培养,L-J绝对浓度间接法进行分枝杆菌的初步菌种鉴定和药物敏感试验。结果 1916株结核分枝杆菌在11种药敏结果中耐一种药物的耐药率为62.8%（1204株）,耐多药（MDR）24.1%（462株）,广泛耐药（XDR）4.0%（76株）。结论重庆市公共卫生医疗救治中心就诊的结核病患者结核分枝杆菌株的总耐药率、MDR和XDR等明显高于全国结核病耐药性基线调查结果,其高耐药趋势与该中心收治基层和周边地区转送难复治结核较多有较密切关系。     

Blood bacteriostatic activity in patients with respiratory tuberculosis]

Vertical diffusion on the L?wenstein-Jensen medium was used to study blood bacteriostatic activity (BBA) in 174 patients with respiratory tuberculosis in relation to the drug resistance of isolated Mycobacterium tuberculosis (MT) strains and to the course of a tuberculous process. There was a clear relationship of BBA to the sensitivity of MT cultures to isoniazid: BAC was high and moderate in 90.8-98.2% of cases if strain drug sensitivity was present and low or null in 98% with drug resistance. The high and medium values of BBA correlated with the clinical indices "improvement" and "significant improvement". Patients with zero and lower values of BBA and isoniazid resistance showed a progressive specific process in 22.0% of cases. Estimation of total BBA by using liquid media is the most informative method.     

Isolation of MPB63 in the culture fluid of Mycobacterium bovis BCG Tokyo: genetical and immunological characterization

MPT63 is a secretory protein first isolated from a culture fluid of M. tuberculosis H37Rv by Nagai et al. In this study, this protein was isolated from an 8-day-culture fluid (Sauton synthetic medium) of M. bovis BCG Tokyo 172 according to Nagai's method. It was shown that M. bovis BCG Tokyo 172 secreted this protein in the medium. The mpt63 gene was detected only in the species of M. tuberculosis complex by polymerase chain reaction (PCR) among 40 different mycobacterial species. Therefore, it is appropriate to designate this protein as MPB63 or MPB/T63 from M. bovis BCG, in similar way as other major secretory proteins of Mycobacteria, such as MPB59 and MPB64. Comparison of the nucleotide sequences of the genes encoding MPB63 protein of M. bovis BCG and MPT63 protein of M. tuberculosis showed only single nucleotide difference at the position 474 where thymine (T) in the former was replaced by adenine (A) in the latter. Amino acid sequences of both proteins were completely identical. MPB63 didn't show delayed-type hypersensitivity (DTH) skin reaction in the sensitized guinea pigs with live or heat-killed M. bovis BCG or heat-killed M. tuberculosis. However, the measurements of serum IgG antibody titers of active tuberculosis patients by enzyme-linked immunosorbent assay (ELISA) showed 74% sensitivity and 96% specificity compared to healthy subjects. Therefore, MPB63 seems to be a promising candidate as an antigen for serodiagnosis of active tuberculosis.     

Pyrazinamidase activity of Mycobacterium tuberculosis--a test of sensitivity to pyrazinamide

Pyrazinamidase activity has been found to correlate with pyrazinamide sensitivity in strains of Mycobacterium tuberculosis. In vitro sensitivity to pyrazinamide in acidified L?wenstein-Jensen medium, and pyrazinamidase activity by the Wayne method, were determined in 378 clinical isolates of M. tuberculosis. A close correlation was observed between the results of both tests. This method of detecting pyrazinamidase activity was found to be a rapid, simple and reliable substitute for pyrazinamide sensitivity testing, and it overcomes the difficulty of growing M. tuberculosis at pH 5.5, as required in the standard method.     

Identification of Mycobacterium bovis among mycobacterial isolates from human clinical specimens at a university hospital in Rio de Janeiro, Brazil

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on L?wenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.     

Serratia marcescens strains implicated in adverse transfusion reactions form biofilms in platelet concentrates and demonstrate reduced detection by automated culture

Background and Objectives Serratia marcescens is a Gram‐negative bacterium that has been implicated in adverse transfusion reactions associated with contaminated platelet concentrates. The aim of this study was to investigate whether the ability of S. marcescens to form surface‐attached aggregates (biofilms) could account for contaminated platelet units being missed during screening by the BacT/ALERT automated culture system. Materials and Methods Seven S. marcescens strains, including biofilm‐positive and biofilm‐negative control strains and five isolates recovered from contaminated platelet concentrates, were grown in enriched Luria–Bertani medium and in platelets. Biofilm formation was examined by staining assay, dislodging experiments and scanning electron microscopy. Clinical strains were also analysed for their ability to evade detection by the BacT/ALERT system. Results All strains exhibited similar growth in medium and platelets. While only the biofilm‐positive control strain formed biofilms in medium, this strain and three clinical isolates associated with transfusion reactions formed biofilms in platelet concentrates. The other two clinical strains, which had been captured during platelet screening by BacT/ALERT, failed to form biofilms in platelets. Biofilm‐forming clinical isolates were approximately three times (P < 0·05) more likely to be missed by BacT/ALERT screening than biofilm‐negative strains. Conclusion S. marcescens strains associated with transfusion reactions form biofilms under platelet storage conditions, and initial biofilm formation correlates with missed detection of contaminated platelet concentrates by the BacT/ALERT system.