利用重组分支杆菌噬菌体快速筛选耐利福平结核分支杆菌

目的：采用基因工程技术构建的可表达荧光素酶的结核分支杆菌噬菌体进行结核分支杆菌利福平药敏试验研究,建立一种特异、灵敏、快速的利福平药敏试验方法。方法采用生物发光方法分别检测重组分支杆菌噬菌体对不同细菌在不同浓度利福平培养其中的发光水平,并与罗氏培养基进行药敏药比较。结果在不同细菌中,噬菌体均特异地对分支杆菌有发光反应,分支杆菌的养基进行药敏试验比较。结果在不同细菌中,噬菌体均特异对对分支杆菌有发光     

应用聚合酶链反应技术检测结核分支杆菌的研究

应用聚合酶链反应技术检测结核分支杆菌的研究谭耀驹李一耕谭守勇我们选用分子量为３８０００片段长度为４１９ｂｐ（３８０００－４１９ｂｐ）系统，对患者的痰液、胸液、脑脊液、支气管冲洗液、淋巴分泌物等进行聚合酶链反应（ＰＣＲ）检测，探讨其在实际应用中的可信性...     

应用多重聚合酶链反应法检测并鉴别结核分支杆菌与非结核分支杆菌DNA

OBJECTIVE: To improve the specificity and sensitivity of polymerase chain reaction (PCR) technique for detecting and identification of DNA of M. tuberculosis and M. nontuberculosis. METHOD: Three pairs of oligonucleotide primer were used in triplex-PCR. A 383 bp DNA fragment encoding part of the 65,000 mycobacterial surface antigen, a 123 bp fragment corresponding to a specific M. tuberculosis complex sequence which was the insertion sequence 6110 (IS 6110) and a 268 bp fragment for human beta-globin were amplified by triplex-PCR respectively. RESULT: The sensitivity of the triplex-PCR-electrophoresis for the DNA of mycobacterium was 0.6 pg. The specific bands of 383 bp and 123 bp among the amplified DNA from M. hominis, M. bovis, BCG and M. simiae were present in the agarose gel. By contrast, only a band of 383 bp was found among the M. nontuberculosis which contained M. avium, M. chelonae, M. scrofulaceum, M. xenopi, M. kansasii, M. intracellulare and M. smegmatis. Compared with the standard strains, there was an additional 268 bp band in simulated clinical samples infected by Mycobacterium. The above 3 specific bands were found neither in other 15 bacterial species tested nor in Mycoplasma pneumoniae. 182 clinical samples were examined by culture, smear and triplex-PCR. 72 nontuberculous clinical samples were all negative. In 110 tuberculosis clinical samples, the positive rates were 2.7%, 13.6% and 32.7%, respectively. CONCLUSION: The triplex-PCR possesses a high specificity and sensitivity. This method could detect and identify the DNA of M. tuberculosis and M. nontuberculosis except M. simiae. It is a valuable tool for early diagnosis and differentiation for infection of M. tuberculosis and M. nontuberculosis.     

结核病人外周血结核分支杆菌聚合酶链反应检测及临床意义

采用巢式聚合酶链反应 (ＰＣＲ)方法扩增结核病人外周血单核细胞 (ＰＢＭＣ)结核分支杆菌 (ＭＴＢ)ＩＳ6 110部分序列 ,并将其克隆与测序 ,结合临床资料分析探讨其临床意义。材料与方法　①对象 :我院 1999年住院的 77例活动性肺结核病人。另以同期住院的非结核病人 (10例肝炎 ,30例肺炎和慢性阻塞性肺疾病 )作对照。②ＰＣＲ引物 :根据Ｔｈｉｅｒｒｙ等[1] 报道的结核分支杆菌ＩＳ6 110序列 ,采用Ｇｏｌｄｋｅｙ软件 ,在其保守区设计两对套式引物 ,委托上海生工生物工程公司合成。引物序列为 :ＴＢ15′ＣＧＴＧＡＧＧＧＣＡＴＣＧＡＧＧＴ…     

利用PCR方法直接从7H12B培养基中鉴定结核分支杆菌的探讨

以ＰＣＲ方法直接从７Ｈ１２Ｂ液体培养基中鉴别结核分支杆菌和非结核分支杆菌，与ＮＡＰ抑制试验对比，具有准确、快速，可应用于临床。     

结核分支村菌耐药分子机制及快速检测的研究

目的：研究结核分支杆菌异烟肼、利福平耐药的分子机制,探索快速检测结核分支杆菌异烟肼、利福平耐药性的分子生物学方法。方法：应用聚合酶反应－单链构象多态性（PCR-SSCP）检测结核分支杆菌异烟肼、利福平耐药株与katG基因、rpoB基因突变。结果：46株结核分支杆菌临床分离株均未发现katG基因序列的缺失,30株异烟肼耐药株中,17株检测到katG基因突变,耐药株中katG基因的突变率为57％;87株结核分支杆菌利福平耐药临床分离株的PCR－SSCP结果显示,所有39株利福平敏感菌无突变检出,48株利福平耐药菌中,36株高度利福平耐药菌和7株低度利福平耐药菌检测量到rpoB基因突变;利福平耐药株中rpoB基因的突变检出率为89．6％。结论：证实katG和rpoB基因突变分别是结核分支杆菌异烟肼和利福平耐的主要分子机制。应用聚合酶链反应－单链构象多态性（PCR－SSCP）可快速检测结核分支杆菌异烟肼、利福平耐药性。     

结核分支杆菌与非结核分支杆菌快速鉴别的实验研究

目的 探讨提供一种从菌种水平快速检测和鉴别分支杆菌的方法。方法 应用三对分别对应于分支杆菌(分子量65000)表面抗原、结核分支杆菌插入序列IS6110及人类β-珠蛋白基因的部分序列的特异性寡核苷酸引物对受试菌种及临床标本中的DNA进行多重PCR扩增，其扩增产物分别为439bp、268bp及123bp，并对439bp进行BstE Ⅱ和Hae Ⅲ两种限制性内切酶消化，同时，将135例临床标本进行培养、涂片，和多重PCR联合限制性片段长度多态性分析(mPCR-RFLP)检测。结果 mPCR-RFLP方法可将12种受试的标准菌种分别区分到种及亚种水平。本方法在临床实验中亦体现出较高的灵敏性及特异性。结论 mPCR-RFLP分析是对结核分支杆菌与非结核分支杆菌进行菌种分类鉴定的一种快速有效的方法。     

Validation of a rapid method for detection of M. tuberculosis resistance to isoniazid and rifampin in Lima, Peru.

SETTING: Treatment of multidrug-resistant tuberculosis (MDR-TB) is often based on drug susceptibility testing (DST) results; for this reason, rapid, simple DST methods are sought which could be applied in resource-poor countries. One such method is a nitrate reductase colorimetric assay known as the Griess method. In Peru, where the incidence rate of TB is among the highest in South America, the National Institute of Health recently undertook the validation and implementation of the direct Griess method. OBJECTIVE: To describe the process of validation and implemention of the direct Griess method at the Peruvian National Institute of Health. DESIGN: Prospective study comparing the sensitivity and specificity of the direct Griess method with the L?wenstein-Jensen proportion method in determining resistance to isoniazid (INH) and rifampin (RMP) among clinical isolates. RESULTS: Among 192 specimens, the sensitivity and specificity of the Griess method for detection of INH resistance was 99.1% and 100%, respectively. For identification of RMP resistance, the sensitivity and specificity was 93.5% and 100%, respectively. CONCLUSIONS: In addition to its high sensitivity and specificity and rapid turn around time, the Griess method uses simple, inexpensive reagents and requires minimal laboratory space and technical expertise, thus providing an ideal screening tool for resource-poor settings with high rates of MDR-TB.     

GeneXpert MTB/RIF for rapid diagnosis and rifampin resistance detection of endobronchial tuberculosis

Background and objective

Delayed diagnosis and treatment of tuberculosis (TB) contribute to poor outcomes, especially for endobronchial TB (EBTB), which typically leads to tracheobronchial stenosis. Finding rapid and accurate diagnostic tools for EBTB is crucial. GeneXpert Mycobacterium tuberculosis (MTB)/rifampin (RIF) was recommended by the World Health Organization (WHO) as a standard molecular biological diagnostic technique for MTB. The aim of this study was to evaluate the efficacy of GeneXpert MTB/RIF for diagnosing EBTB and for evaluating RIF resistance.

Methods

Biopsy tissue and bronchial brushings from EBTB patients were prospectively assessed with GeneXpert MTB/RIF. The diagnostic yields of auramine O‐stained sputum smears and bronchial brush smears were obtained, and the results were compared with the cultures of sputum and biopsy tissues for MTB.

Results

In 61 confirmed cases of EBTB, the sensitivities of sputum smear, bronchial brush smear, sputum culture and tissue culture to diagnose EBTB were 13.1%, 32.8%, 36.1% and 68.9%, respectively. For bronchial brushings and biopsies, our data showed sensitivities of 57.4% and 63.9%, respectively, and a specificity of 100% for GeneXpert MTB/RIF, and these results were superior to those of sputum smears, bronchial brush smears and sputum culture. GeneXpert MTB/RIF for bronchial brushings and biopsies showed complementarity in its diagnostic performance. Resistance to RIF was identified in 17.4% (8/46) of GeneXpert MTB‐positive cases.

Conclusion

GeneXpert MTB/RIF may enable more rapid EBTB diagnosis and determination of RIF resistance, which are crucial for timely treatment.

     

Primary drug-resistant tuberculosis in children. Emergence of primary drug-resistant strains of M. tuberculosis to rifampin

A prospective study of primary drug-resistant strains of Mycobacterium tuberculosis among children was begun at the Kings County Hospital Medical Center of Brooklyn in 1961 and reported at 5 4-yr periods through 1980. The present report extends our observations of primary drug-resistant tuberculosis in children through 1984. The salient finding in the present report was the increase in primary drug resistance to rifampin, 3 of 19 strains resistant in the last period of study (1981 to 1984) as compared with 1 of 96 strains isolated in the previous 3 periods of study (1969 to 1980). This increase was significant (p less than 0.02) even though the number of strains isolated was small. There were continued low resistance rates to ethambutol and para-aminosalicylic acid and stable resistance rates for isoniazid and streptomycin.     

结核分枝杆菌及利福平耐药核酸扩增检测成本分析

目的 比较固体培养、比例法药敏和结核分枝杆菌及利福平耐药核酸扩增（Xpert Mtb/RIF）(简称“Xpert”)检测在中国基层实验室应用的检测成本。 方法 选取4个县（区）级结核病防治机构收集3次固体培养和Xpert检测成本数据,2个省参比实验室收集3次对硝基苯甲酸（PNB）菌群鉴定和比例法利福平药敏试验成本数据。采用要素法计算每种方法单位检测成本,计算每例患者不同检测方法的检测成本。在不同结核病患病率人群中,以固体培养试验为金标准,比较Xpert检测在不同检测效能下检出1例结核病患者的成本。在不同利福平耐药率的可疑人群中,以传统药敏试验为金标准,比较Xpert检测在不同检测效能下检出1例利福平耐药结核病患者的成本。调整Xpert检测设备和试剂价格后,分析Xpert检测的单位成本。 结果 固体培养、菌群鉴定、利福平药敏试验和Xpert检测的单位检测成本分别为47.87、46.73、82.86和118.62元。传统方法检测每例结核病患者所需成本（2份培养,1份传代,1份PNB鉴定试验）为172.57元。传统方法检测每例耐药结核病患者的平均成本（2份固体培养,1份传代,1份PNB鉴定及药敏试验）为208.84元。当Xpert检测结核病或利福平耐药特异度为85%时,如果检测敏感度大于70%,在结核病患病率或利福平耐药结核病患病率1%～70%的任何人群中,Xpert检出1例结核病或利福平耐药结核病患者的成本均低于传统方法。试剂价格变化对于检测成本的影响远远大于设备价格变化的影响。 结论Xpert检测是一种比传统方法更经济并快速的检测方法,适合在中国基层实验室推广。     

197例临床结核杆菌分离株的快速耐药性检测报告

目的评估线性探针MTBDR-Plus检测临床分离株对利福平（RFP)和异烟肼（INH)药物敏感性的效能。方法应用病例对照研究方法选取197例临床结核菌分离株,进行传统比例法药物敏感性试验和MTBDR-Plus检测,分析MTBDR-Plus方法的敏感性和特异性。结果 MTBDR-Plus方法检测RFP和INH耐药的敏感性分别为88.6%和79.3%,特异性分别为98. 8%和98.8%。结论MTBDR-Plus方法灵敏度和特异性高,具有较好的应用前景。     

Development of a pyrosequencing approach for rapid screening of rifampin, isoniazid and ethambutol-resistant Mycobacterium tuberculosis.

SETTING: The need to minimize the transmission of drug-resistant Mycobacterium tuberculosis requires rapid identification procedures. OBJECTIVE: To develop a pyrosequencing approach for rapid screening of rifampin, isoniazid and ethambutol-resistant M. tuberculosis based on characterization of resistance-associated hot mutations. DESIGN: Three pairs of PCR primers and three pyrosequencing sequencing primers for detecting mutations at codon 526 and 531 of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene were chosen. The sensitivity of the pyrosequencing approach was determined by assaying PCR products generated from 10-fold serial dilutions of the DNA from the H37Rv strain. The efficacy of the pyrosequencing approach was evaluated by analyzing clinical isolates with a known antibiotic phenotype. RESULTS: Resistance-associated hot mutations could be determined within 2 h after PCR amplification using pyrosequencing. About 45 fg DNA per reaction was required to obtain sufficient PCR products to produce a clear, accurate pyrosequencing pattern. No mutations were found in all 20 drug-susceptible clinical isolates, while all isolates with mutations showed corresponding drug resistances. CONCLUSION: This pyrosequencing approach can be used for rapid screening of rifampin-, isoniazid- and ethambutol-resistant M. tuberculosis prior to standard drug susceptibility testing.     

结核分支杆菌耐利福平与rpoB基因

90年代初 ,全球结核病呈现死灰复燃趋势。到目前为止 ,结核病仍是造成成人死亡的感染性疾病中最常见的病因 ,全球 2 6 %的可避免死亡与结核病有关。我国约有 6 0 0万结核病患者 ,每年死于结核病者约 2 5万 ,为死于其他各类传染病人数总和的 2倍 ,每年由结核病带来的直接损失超过 35亿。结核病疫情恶化的原因主要有两方面 :一是耐药 ,尤其是耐多药结核 (ＭＤＲ ＴＢ)的出现 ;另一原因是人类免疫缺陷病毒 (ＨＩＶ)感染的爆发流行。我国结核病具有耐药率高的特点 ,初治病人中耐药结核病占 2 8 1% ,复治病人中占 41 1%。耐药结核病中继发耐药占…     

Evaluation of the resazurin microtiter assay for rapid detection of ofloxacin resistance in M. tuberculosis.

OBJECTIVE: To evaluate the performance of the colorimetric resazurin microtiter assay (REMA) method for the detection of ofloxacin resistance. METHODS: A panel of 120 multidrug-resistant Mycobacterium tuberculosis strains was tested blindly by the REMA method and compared with the results obtained using the BACTEC 460 method. RESULT: A very good correlation was observed between the two methods. CONCLUSION: The REMA method is simple, rapid and can be an inexpensive alternative procedure for the rapid detection of anti-tuberculosis drug resistance in laboratories with limited resources.