Fast and Accurate Identification of <i>M. tuberculosis</i>Complex Using an Immunochromatographic MPT64 Antigen Detection Test

Background: A new rapid Immunochromatographic test (ICT) kit (MPT64 TB Ag Kit) for detection of MPT64 Antigen in M. tuberculosis (MTB) isolates used for rapid identification of MTB isolates developed by SD (Standard Diagnostics) Bio line, South Korea was evaluated. The ICT is a rapid, reliable and cheaper method that can be used instead of conventional biochemical tests for confirming MTB in culture isolates in resource limited laboratories. The study also evaluated the ability of ICT to detect MPT64-Antigen before the micro MGIT could signal positive. Material/Methods: A total of 450 sputum samples of individual patients were used for the study. 152 isolates of Mycobacteria were recovered from solid and liquid media. These strains were tested for the detection of MPT64-antigen. H37Rv strain was served as the positive reference control and also used for early detection of Antigen experiment. Findings: The development of bands on both test and sample region when H37Rv strain was tested were seen (MPT64 antigen positive). When 138 MTB isolates were tested, it showed a similar banding pattern indicating 100% sensitivity. MPT64 band formation was not detected in any of the 14 isolates indicating 100% specificity. Both PPV & NPV were 100%. All the isolates negative for MPT64 Ag were confirmed as MOTT by conventional bio-chemical PNBA. The H37Rv strain showed a faint band from the 2nd day onwards from inoculation till 3rd day in the earlier Antigen detection experiment. Conclusion: Rapid identification of MTB culture isolate is a pressing need for diagnosis and proceeding to perform drug susceptibility testing. MPT64 TB Ag detection ICT kit is a rapid, reliable method, good substitute for molecular identification methods, and conventional biochemical test which is time-consuming and technically demanding. The early detection of Antigen can be used as an effective tool in diagnosis.

Application of PCR-SSCP in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis

Objective: To study the relationship between the polymorphism of drug resistant gene rpoB and drug resistance against rifampicin(RFP) of M. tuberculosis L-forms, and to evaluate its clinical application. Methods: A total of 52 clinical isolated strains of M. tuberculosis L-forms were collected. rpoB gene polymorphism was analyzed by polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) and conventional antimicrobial susceptibility test (AST). Their results were compared. Results: AST results showed that 38 of 52 clinical isolated strains were drug resistance (73.08％),while PCR-SSCP indicated 65.38％ (32/52) rpoB gene polymorphism. There was no statistic significance(χ2= 2.4914) between the 2 methods. Conclusion:Combined the application of PCR-SSCP with AST in detecting rpoB drug resistant gene polymorphism of M. tuberculosis L-form from pneumoconiosis patients with tuberculosis may have advantages at earlier diagnosis and guidance of clinical medications.

A new Multilocus Sequence Analysis Scheme for Mycobacterium tuberculosis

Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis （M. tuberculosis） multilocus sequence analysis （MLSA） was established for the phylogenetic and epidemiology analysis. Methods To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods. Results After comparison of the genome, seven high discrimination gene loci （recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR） were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types （STs） were identified. Based on the Hunter-Gaston Index （HGI）, MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types （ST） came into a main cluster, showing the major clonal complexes in those 44 strains. Conclusion MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

First Line Anti-Tuberculosis Drugs Resistance Patterns of <i>Mycobacterium tuberculosis</i>Isolates from Newly Diagnosed Cases of Tuberculosis

Introduction: Tuberculosis is a major cause of mortality and morbidity world-wide. Anti-tuberculosis drugs have been used for many decades but resistance to them is now widespread. Globally 5% of tuberculosis cases and in India 3% among new TB cases. This study was planned to know the pattern of first line anti-tuberculosis drug resistance in south Gujarat, Surat region in newly diagnosed patients of tuberculosis. Material and Methods: 350 samples were processed for homogenisation and concentration using 4% NAOH-2.9% trisodium citrate. Processed samples were inoculated in liquid medium that is MGIT (Mycobacterial growth indicator tube). Positive samples for M. tbwere processed further for first line anti-tuberculosis drugs sensitivity testing (DST). Reading was taken by using MicroMGIT system. Result: Out of 350 samples 59 (17%) were positive samples, of which 48 (13%) were M. tb and 11 (3%) were non tuberculous mycobacteria. Out of 48 samples 2% (1 isolate) was resistant to isoniazid and Rifampicin while 2% were monoresistant to isoniazide, 2% monoresistant to streptomycin. No rifampicin monoresistant was detected. Conclusion: Such study may help in control of tuberculosis at regional and national level which would in turn help in planning of measures to control Multi-drug resistance tuberculosis. Continuous surveillance should be applied to know the periodic changing patterns and trend in Drug resistant tuberculosis.

Evaluation of Recombinant <i>Mycobacterium tuberculosis</i>Antigens MPT64, CFP10, and ESAT6 for Delayed-Type Hypersensitivity Responses in Guinea Pigs

The tuberculin Purified Protein Derivative (PPD) skin test is widely used;however, the results are often inaccurate. Positive results can be observed in patients with active tuberculosis (TB) as well as in BCG-vaccinated persons and individuals who are infected with mycobacteria but have not developed the disease. MPT64, an antigen secreted from actively growing Mycobacterium tuberculosis and some strains of M. bovis BCG such as BCG Tokyo and BCG Russia, is immunogenic and elicits delayed-type hypersensitivity (DTH) in guinea pigs and humans. This antigen has been used to develop a new skin test for the diagnosis of active TB infection. Two of the antigens encoded by the M. tuberculosis-specific region of difference 1 (RD1, deleted in M. bovis BCG strains), CFP10 (culture filtrate protein 10) and ESAT6 (early secreted antigenic target-6), also induce M. tuberculosis-specific DTH responses. The aim of this study was to investigate the DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo elicited by three purified recombinant proteins (rMPT64, rCFP10 and rESAT6) compared to those elicited by PPD. In this study genes encoding MPT64, CFP10, and ESAT6 were cloned and expressed as recombinant proteins with the addition of a C-terminal His6 tag for ease of purification by Immobilized Metal ion Affinity Chromatography (IMAC). The recombinant proteins (rMPT64, rCFP10, and rESAT6) were purified to homogeneity and were used to elicit DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo. The results showed that rMPT64 elicits a DTH response comparable to that of PPD in M. bovis BCG Tokyo-vaccinated animals. However, M. tuberculosis-infected animals show less reactivity to rMPT64 than they do to PPD. Although single rCFP10 or rESAT6 did not readily elicit a DTH response in M. tuberculosis-infected animals, combining these antigens with rMPT64 led to an increased DTH response, thus enabling the detection of TB infection.

Estimates of Genetic Variability of <i>Mycobacterium tuberculosis</i>Complex and Its Association with Drug Resistance in Cameroon

The present study investigates the genetic diversity among Mycobacterium tuberculosis complex circulating in the Centre region of Cameroon and analyzes the relationship between genotypes and drug resistance patterns. Spoligotyping was performed by PCR-amplification followed by the reverse hybridization of 298 cultured specimens. Spoligotypes patterns were identified by comparison to reference strains in SPolDB4 database via the MIRU VNTR plus web application. About 97.65% of all tuberculosis (TB) cases were attributed to M. tuberculosis. A total of 65 different profiles were identified. Of these, 40 were represented as Shared Types (ST) while the others were orphans. LAM10_CAM and Haarlem families were the most prevalent genetic families with 51.01% and 14.09% respectively. ST 61, a member of the LAM10_ CAM family formed the largest cluster with 128 (42.95%) isolates. No association was found between genotypes with regard to drug resistance and HIV sero-status. However, there was a significant association between genotypes and age groups. Patients belonging to 15 - 24 and 35 - 44 age groups were more likely infected by LAM10_CAM strains compared to others. The population structure of Mycobacterium tuberculosis complex strains from the Centre region was found to be diverse and the spoligotype 61 of the LAM10_CAM family was highly predominant. Isolates of the LAM10_CAM seem to be not associated with drug resistance.

VNTR及RD105缺失检测法对新疆石河子市汉族MTB基因分型及其耐药的研究

了解新疆石河子地区汉族结核分枝杆菌基因型特征、"北京家族"基因型的分布及其与耐药的相关性。应用多位点可变串联重复序列(VNTR)技术、RD105缺失检测法分析结核分枝杆菌的基因型,聚类分析采用Bionum-berics5.0软件分析。55株结核分枝杆菌临床分离株中,"北京家族"基因型47株;24位点VNTR检测结果显示明显的基因多态性,经基因聚类分析,可分为7个基因簇和46个独立的基因型,分为8个基因群(Ⅰ-Ⅷ群),其中46株属于Ⅵ群;耐药菌株为19株(34.5%)。新疆石河子地区结核分枝杆菌有明显的基因多态性;"北京家族"基因型为主要流行株,应引起重视;"北京家族"基因型与耐药无明显相关性。

论“εrdεm”主题及其在高昌回鹘文学中的表现

文章通过道德这一题材在高昌回鹘文学中的表现,指出它与人、社会及法律的密切关系。依据大量实例,探讨了“道德”主题在高昌回鹘文学中的风俗、科技、宗教信仰、自然、环境、爱国主义、人道主义、集体主义等内容的表现形式。

Screening of Several Anti-Infectives for in Vitro Activity against Mycobacterium smegmatis

Aim: To evaluate in vitro the effectiveness of several anti-infective agents alone or in combination against Mycobacterium smegmatis. Method: A convenient stratified sampling method was used to obtain selected anti-infective agents. For individual drug samples, Minimum Inhibitory Concentrations (MIC) were obtained using the agar-well plate diffusion technique. Fractional Inhibitory Concentration Indices (FICI) were calculated for drug combinations using their MIC as obtained from the broth dilution method. Results: Of the thirty (30) anti-infective agents analyzed, ten (10) had MIC equivalent to or better than rifampicin (reference TB drug). Seven (7) drugs had MIC higher than rifampicin, while twelve (12) showed no growth inhibition of M. smegmatis. Analysis of the effect of drug combinations on M. smegmatis indicated that four (4) combinations, including rifampicin/ethambutol showed synergism. One (1) was additive, two (2) were indifferent and one (1) combination showed antagonism. Conclusion: Notable in the results obtained was the high effectiveness of the carbapenems in inhibiting the growth of M. smegmatis. Carbapenems, though not indicated for TB treatment, has a potential of playing a significant role in the treatment of tuberculosis. Also the drug combinations which showed synergism, especially those that involved the macrolide antibiotics, should further be investigated. These results have to be confirmed by in vivo clinical studies to define their roles in tuberculosis treatment.

Evaluation of Increased Sensitivity of Morning Bleach Sample for Detection of Acid Fast Bacilli in Pulmonary Samples

Around 85% of the cases of Tuberculosis (TB) are pulmonary in origin and the routine diagnosis usually depends on sputum microscopy. The conventional direct Ziehl-Nelson (ZN) staining technique has been found to have a low sensitivity. The main objective of the study was to verify whether the bleach concentration method increases the sensitivity of sputum smear microscopy for AFB or not and also to see whether the first single morning sample alone is sufficient and better than the three pooled samples after bleach concentration followed by ZN staining. A total of 365 samples were studied from 131 clinically suspected cases of pulmonary TB which included sputum (112), gastric aspirate (5), endotracheal tube washing (2), and bronchial lavage (12). All these samples were processed for conventional ZN staining and Bleach concentration method followed by ZN staining. An increase in positivity was observed in all the cases after using the bleach concentration method and the most significantly useful was that in the case of first morning sputum samples where it increased from 11.6% to 41.96%. Bleach concentration is a simple, cheap and easily available method and also very safe because it kills the Mycobacteria in the process. Its positivity rate is better as compared to direct conventional ZN staining.

Taq Man-PCR技术诊断结核病的临床应用研究

目的 探讨TaqMan -聚合酶链反应 (TaqMan -PCR)技术在结核病快速诊断中的临床价值。方法 对 15 5例活动性肺结核患者的痰和外周血、130例结核性胸膜炎患者的胸腔积液和外周血以及 6 1例结核性脑膜炎患者的脑脊液和外周血应用TaqMan -PCR检测结核分支杆菌DNA ,痰、胸腔积液和脑脊液进行抗酸染色涂片检查 ,另外 ,痰标本还进行了BACTEC和改良罗氏培养 ;同期以5 2例肺癌患者的痰和外周血、5 0例恶性胸腔积液患者的胸腔积液以及 33例健康人的外周血作为对照进行TaqMan -PCR检测。 结果 15 5例活动性肺结核患者的痰和外周血、130例结核性胸膜炎患者的胸腔积液和外周血以及 6 1例结核性脑膜炎患者的脑脊液和外周血TaqMan -PCR的阳性率分别为 4 9.0 %和 5 1.6 %、4 5 .4 %和 38.5 %以及 5 0 .8%和 4 2 .6 % ,痰、胸腔积液和脑脊液TaqMan -PCR的阳性率显著高于抗酸染色涂片以及BACTEC和罗氏培养 (P <0 .0 5 )。TaqMan -PCR检测痰、胸腔积液和外周血的特异性分别为 96 .2 %、98%和 96 .5 %。结论 TaqMan -PCR具有较高的敏感性和特异性 ,对结核病的快速诊断具有一定的价值。

结核杆菌含信号肽的Mtb8.4真核表达质粒的构建及鉴定

目的 对结核杆菌新抗原—带信号肽的Mtb8.4(MS)进行基因克隆 ,构建其真核表达质粒并加以鉴定。 方法 采用聚合酶链反应 (PCR)从结核分枝杆菌H 3 7Rv株基因组中扩增出带信号肽的Mtb8.4(MS)目的基因 ,经HindⅢ和EcoRⅠ消化后 ,与 pcDNA3 .1(+ )载体进行连接重组。 结果 pcDNA3 .1(+ ) -MS真核表达质粒构建完成后 ,用限制性内切酶消化、PCR及DNA测序等多种方法进行鉴定 ,证实其构建成功。 结论 pcDNA3 .1(+ ) -MS真核表达质粒的成功构建 ,为进一步研究该质粒的免疫保护效果 ,了解信号肽序列在MS蛋白表达和分泌过程中所起的作用及制备相应的结核病DNA疫苗奠定了基础。

噬菌体TM4复苏结核休眠菌的初步研究

目的探索噬菌体TM4对结核休眠菌复苏的作用。方法采用密闭培养对数生长期结核菌构建休眠菌模型;采用药敏检测和透射电子显微镜(电镜)观察作为模型检测方法;采用菌落计数和电镜观察作为复苏的检测指标。结果密闭培养180 d休眠菌模型构建成功;培养3 d复苏促进因子(Rpf)E组管底菌量多于空白组和TM4组,培养8 d噬菌体TM4组管底菌量多于空白组,后续观察见TM4组和Rpf E组管底菌量均较前有所增加。混合液培养第1日时,空白组、TM4组和Rpf E组的菌落计数均为0;培养第6日时,菌落计数分别是0、0.7×102和2×104CFU/mL;培养第14日时,菌落计数分别是3.4×10、1.68×107和2.1×109CFU/mL。培养第17日时,电镜观察发现混合物TM4组有大量薄壁结核菌、部分厚壁结核休眠菌和结核菌细胞碎片,Rpf E组满视野薄壁的结核菌,空白组含大量厚壁的结核休眠菌和少数薄壁结核菌。结论噬菌体TM4能够复苏结核休眠菌。

肺结核患者血浆和胸水中抑瘤素-M检测及临床意义

目的探讨血浆和胸水中抑瘤素-M在肺结核中的变化及其临床意义。方法应用双抗体夹心ELISA法检测24例健康体检者、21例活动性肺结核患者血浆和10例结核性胸膜炎胸水中OSM的水平。结果肺结核患者血浆中OSM水平低于健康对照组,差异有统计意义;结核性胸膜炎患者胸水中OSM高于肺结核患者血浆中OSM水平,差异有统计意义。结论OSM是局部病灶炎症反应的重要作用因子,可能参与了肺结核的免疫发病机制。

乙醇对结核分枝杆菌M_r 38000蛋白在大肠杆菌中可溶性表达的促进作用

目的:探讨乙醇促进结核分枝杆菌Mr38000蛋白 的可溶性表达,并获得可溶性的Mr38000重组蛋白.方法: 采用聚合酶链反应(PCR)从结核分枝杆菌H37Rv基因组中 扩增出Mr38000蛋白的编码基因,用限制性内切酶消化后插 入载体pGEX 4T 2中,将重组质粒转化大肠杆菌BL21,在乙 醇添加剂存在下,经IPTG诱导,表达GST 38融合蛋白;聚丙 烯酰胺凝胶电泳(SDS PAGE)分析融合蛋白的相对分子质量 及表达形式,免疫印迹法鉴定融合蛋白的活性.结果:经PCR 扩增后证实为Mr38000蛋白的基因,成功构建了具有正确基 因序列的重组表达载体,在大肠杆菌BL21中诱导表达融合蛋 白约占菌体总蛋白的18%.在乙醇存在下,可溶性目的蛋白的 表达量约是无乙醇时的5倍.结论:在原核表达中乙醇作为一 种添加剂可以促进结核分枝杆菌Mr38000蛋白在大肠杆菌 BL21中的可溶性表达.

结核杆菌含信号肽的Mtb8.4真核表达质粒的构建及在COS-7细胞中的表达

目的:克隆结核杆菌含信号肽的Mtb8·4(MS)基因,导入真核表达载体pcDNA3·1(+),构建成重组质粒pcDNA3·1(+)-MS,并在真核细胞中进行表达。方法:提取人结核杆菌H37Rv株的基因组作为模板,进行PCR圹增,获得含信号肽的Mtb8·4(MS)基因后,与pcDNA3·1(+)载体进行连接重组,用限制性内切酶消化、PCR及DNA序列分析等多种方法进行鉴定;重组质粒转染COS-7细胞48h后,用RT-PCR方法鉴定MS在转录水平的表达情况。结果:pcD-NA3·1(+)-MS真核表达载体构建成功;转染COS-7细胞后,MS在转录水平成功表达。结论:pcDNA3·1(+)-MS真核表达质粒的构建以及MS在COS-7细胞中的成功表达,为进一步研究该真核表达质粒的免疫保护效果及制备结核病pcD-NA3·1(+)-MSDNA疫苗奠定了基础。

结核分枝杆菌新抗原Mtb8.4基因的克隆与真核表达

目的克隆结核杆菌新抗原Mtb8.4基因,导入真核表达载体pcDNA3.1(+),构建成重组质粒pcDNA3.1(+)-Mtb8.4,并在真核细胞中进行表达。方法提取人结核杆菌H37Rv株的基因组作为模板,进行PCR扩增,获得Mtb8.4目的基因后,与pcDNA3.1(+)载体进行连接重组,用限制性内切酶消化、PCR及DNA序列分析等多种方法进行鉴定;重组质粒转染COS-7细胞48h后,用RT-PCR方法鉴定Mtb8.4在转录水平的表达情况。结果pcDNA3.1(+)-Mtb8.4真核表达载体构建成功;转染COS-7细胞后,Mtb8.4在转录水平成功表达。结论pcDNA3.1(+)-Mtb8.4真核表达质粒的构建成功以及Mtb8.4在COS-7细胞中的成功表达,为进一步研究该真核表达质粒的免疫保护效果及制备结核病pcDNA3.1(+)-Mtb8.4DNA疫苗奠定了基础。

结核分枝杆菌稳定L型耐乙胺丁醇embB基因的研究

目的探讨结核分枝杆菌L型形成乙胺丁醇耐药性的分子机制。方法采用非高渗分离培养法在不含乙胺丁醇与含乙胺丁醇的液体培养基内诱导形成结核分枝杆菌L型,过滤传代后获得稳定L型纯培养物。通过nPCR扩增、PCR-SSCP及PCR-DS技术分别对自发形成与诱导形成的结核分枝杆菌稳定L型耐乙胺丁醇embB基因进行检测和分析。结果自发形成及药物诱导形成的结核分枝杆菌稳定L型embB基因的nPCR及PCR-SSCP结果显示,与其亲代细菌型一致的DNA条带和带型;测序结果显示稳定L型embB基因序列与其亲代细菌相同没有发生改变,其结果与PCR-SSCP分析结果一致。结论 PCR-SSCP和PCR-DS技术可用于结核分枝杆菌L型耐乙胺丁醇基因的检测;无论是自发形成或药物诱导形成的稳定L型embB基因未发生突变,提示结核分枝杆菌L型对乙胺丁醇的耐药性同embB基因突变无关,细胞壁缺陷变异可能是导致结核分枝杆菌产生乙胺丁醇耐药性的一个重要机制。

结核分枝杆菌含信号肽的Mtb8.4基因的克隆及真核表达质粒的构建(英文)

目的对结核杆菌新抗原—带信号肽的Mtb8.4(MS)进行基因克隆,构建其真核表达质粒并加以鉴定。方法采用聚合酶链反应(PCR)从结核分枝杆菌H37Rv株基因组中扩增出带信号肽的Mtb8.4(MS)目的基因,经HindⅢ和EcoRⅠ消化后,与pcDNA3.1(+)载体进行连接重组。结果pcDNA3.1(+)-MS真核表达质粒构建完成后,用限制性内切酶消化、PCR及DNA测序等多种方法进行鉴定,证实其构建成功。结论pcDNA3.1(+)-MS真核表达质粒的成功构建,为进一步研究该质粒的免疫保护效果,了解信号肽序列在MS蛋白表达和分泌过程中所起的作用及制备相应的结核病DNA疫苗奠定了基础。

HBV-M阳性肺结核患者抗结核化疗的探讨

探讨ＨＢＶ－Ｍ阳性肺结核患者抗结核化疗对肝功能的影响。方法：采用酶联免疫吸附试验（ＥＬＩＳＡ）法检测１７８例肺结核患者ＨＢＶ－Ｍ、观察执结核化疗中肝功能变化与ＨＢＶ－Ｍ的关系。结果：１７８例肺结核患者中ＨＢＶ－Ｍ阳性９２例（５１．７％）、阴性８６例（４８．３％）、化疗期间肝功能异常分别为３４．８％和９．４％（Ｐ＜０．００１）。单阳祖、双阳组和三阳组肝功能异常分别为２３．１％，４４．４％和５７．７％，三阳组显著高于单阳组和双阳组（Ｐ＜０．０５）、结论ＨＢＶ－Ｍ阳性肺结核患者可用含ＩＮＨ、ＲＦＰ和Ｐ２Ａ方案，但应并用保肝药。ＨＢＳＡｇ（＋）、ＨＢｅＡｇ（＋）、ＨＢＣＡｂ（＋）模式最好不用Ｐ２Ａ。