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 ...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.展开更多
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 D...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.展开更多
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 to...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.展开更多
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 an...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.展开更多
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-vaccina...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.展开更多
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. ...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.展开更多
目的系统评价免疫相关鸟苷三磷酸酶(IRGM)基因rs10065172位点多态性与结核病发病风险的相关性。方法通过计算机检索PubMed、万方全文数据库、中国期刊全文数据库(CNKI)、中国生物医学文献数据库(CBM)、维普数据库等数据库,查找关于IRGM...目的系统评价免疫相关鸟苷三磷酸酶(IRGM)基因rs10065172位点多态性与结核病发病风险的相关性。方法通过计算机检索PubMed、万方全文数据库、中国期刊全文数据库(CNKI)、中国生物医学文献数据库(CBM)、维普数据库等数据库,查找关于IRGM基因rs10065172位点多态性与结核病发病风险相关性的病例对照研究,检索时限为各个数据库建库至2017年3月。由两名评价员独立对文献进行筛选及数据提取,运用RevMan 5.3和Stata 12.0软件进行Meta分析。结果共纳入文献8篇,9个病例对照研究,包括3 956例结核病病人,4 896例健康对照。Meta分析结果显示,在总体人群合并分析中,IRGM基因rs10065172位点多态性与结核病之间的相关性在隐性基因模型(TT vs TC+CC:OR=0.87,95%CI=0.78~0.97,P=0.01)、共显性基因模型(TT vs CC:OR=0.89,95%CI=0.78~1.01,P=0.08;TT vs TC:OR=0.85,95%CI=0.76~0.96,P=0.007)中有统计学意义。在亚洲人群中,IRGM基因rs10065172位点多态性与结核病之间的相关性在共显性基因模型(TT vs CC:OR=0.80,95%CI=0.67~0.95,P=0.01;TT vs TC:OR=0.80,95%CI=0.68~0.94,P=0.007)、等位基因模型(T vs C:OR=0.91,95%CI=0.84~0.99,P=0.03)、隐性基因模型(TT vs TC+CC:OR=0.80,95%CI=0.69~0.93,P=0.005)中均具有统计学意义。高加索人群及非洲人群因纳入文献较少,未发现IRGM基因多态性与结核病发病风险之间的相关性。结论在亚洲人群中IRGM基因rs10065172位点多态性与结核病发病风险有相关性,其中野生型等位基因C与结核病发病风险增高相关。展开更多
基金supported by the fund of State Key Laboratory for Infectious Diseases Prevention and Control (2011SKLID208)the project "Transmission Mode of Tuberculosis"of National Key Program of Mega Infectious Diseases (2008ZX100/03-010)
文摘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.
文摘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.
文摘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.
文摘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.
文摘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.
文摘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.
文摘目的系统评价免疫相关鸟苷三磷酸酶(IRGM)基因rs10065172位点多态性与结核病发病风险的相关性。方法通过计算机检索PubMed、万方全文数据库、中国期刊全文数据库(CNKI)、中国生物医学文献数据库(CBM)、维普数据库等数据库,查找关于IRGM基因rs10065172位点多态性与结核病发病风险相关性的病例对照研究,检索时限为各个数据库建库至2017年3月。由两名评价员独立对文献进行筛选及数据提取,运用RevMan 5.3和Stata 12.0软件进行Meta分析。结果共纳入文献8篇,9个病例对照研究,包括3 956例结核病病人,4 896例健康对照。Meta分析结果显示,在总体人群合并分析中,IRGM基因rs10065172位点多态性与结核病之间的相关性在隐性基因模型(TT vs TC+CC:OR=0.87,95%CI=0.78~0.97,P=0.01)、共显性基因模型(TT vs CC:OR=0.89,95%CI=0.78~1.01,P=0.08;TT vs TC:OR=0.85,95%CI=0.76~0.96,P=0.007)中有统计学意义。在亚洲人群中,IRGM基因rs10065172位点多态性与结核病之间的相关性在共显性基因模型(TT vs CC:OR=0.80,95%CI=0.67~0.95,P=0.01;TT vs TC:OR=0.80,95%CI=0.68~0.94,P=0.007)、等位基因模型(T vs C:OR=0.91,95%CI=0.84~0.99,P=0.03)、隐性基因模型(TT vs TC+CC:OR=0.80,95%CI=0.69~0.93,P=0.005)中均具有统计学意义。高加索人群及非洲人群因纳入文献较少,未发现IRGM基因多态性与结核病发病风险之间的相关性。结论在亚洲人群中IRGM基因rs10065172位点多态性与结核病发病风险有相关性,其中野生型等位基因C与结核病发病风险增高相关。