Objective A PCR-reverse dot blot hybridization （RDBH） assay was developed for rapid detection of rpoB gene mutations in ＇hot mutation region＇ of Mycobacterium tuberculosis （M. tuberculosis）. Methods 12 oligonucl...Objective A PCR-reverse dot blot hybridization （RDBH） assay was developed for rapid detection of rpoB gene mutations in ＇hot mutation region＇ of Mycobacterium tuberculosis （M. tuberculosis）. Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing （DST） and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2% （165/181） and 98.3% （117/119）, respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value （PPV） and negative predictive value （NPV） of the RDBH assay were 97.7% （293/300）, 98.2% （164/167）, and 97.0% （129/133）, respectively. Furthermore, the results indicated that the most common mutations were in codons 531 （48.6%）, 526 （25.4%）, 516 （8.8%）, and 511 （6.6%）, and the combinative mutation rate was 15 （8.3%）. One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.展开更多
Background China is one of the high burden countries of Mycobacterium tuberculosis （TB） infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin （RIF） and isoniaz...Background China is one of the high burden countries of Mycobacterium tuberculosis （TB） infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin （RIF） and isoniazid （INH） resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance.Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide （RIFO） assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA （-15C/T） and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism （RFLP） method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups. Results Sixty-five （91.5%） of 71 RIF resistant and 52 （92.9%） of 56 multidrug-resistant （MDR, i.e. resistant to at least RIF and INH） strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively, katG315 AGC〉ACC and inhA-15C〉T mutations were found in 40 （60.6%） and 10 （15.2%） of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into ＂modern＂ （76.6%） and ＂ancestral＂ （23.4%） group. There is no significant difference between ＂ancestral＂ and ＂modern＂ group in prevalance of drug resistance-associated gene mutations. Conclusions The hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains; a combined use of both katG315 and inhA-15 can improve the detection rate of I NH resistant strains; the Beijing genotype is prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.展开更多
Background The Xpert MTB/RIF showed high sensitivity and specificity in previous studies carried out in different epidemiological and geographical settings and patient populations in high-burden tuberculosis （TB） co...Background The Xpert MTB/RIF showed high sensitivity and specificity in previous studies carried out in different epidemiological and geographical settings and patient populations in high-burden tuberculosis （TB） countries.However,there were little data obtained by validation or demonstration study of the assay in China.In this study,the performance of Xpert MTB/RIF was investigated in two county-level laboratories in Hunan Province,China.Methods Consecutive patients with suspected pulmonary tuberculosis （PTB） and suspicion for multidrug-resistant tuberculosis （MDR-TB） were enrolled.For each patient suspected to have PTB,three sputum specimens （one spot sputum,one night sputum,and one morning sputum） were collected and each sputum was tested with smear microscopy,L（o）wenstein-Jensen （LJ） culture,and Xpert MTB/RIF test.For comparison across subgroups and testing methods,95％ confidence intervals were calculated.All analyses were done with SPSS 16.0,and P ＜0.05 was regarded as significant.Results For case detection,the sensitivity of Xpert MTB/RIF was 100％ for smear-and culture-positive TB and 88.6％ for smear-negative and culture-positive TB; the overall sensitivity was 94.5％ for all culture-positive patients.The specificity was 99.8％.The sensitivity of Xpert MTB/RIF assay was 22.0％ in clinical TB patients and the specificity reached 100.0％ in the group of patients who are infected with nontuberculous mycobacteria.For the detection of rifampin resistance,the sensitivity of MTB/RIF RIF-resistance detection was 92.9％,and the specificity was 98.7％.Of the 26 Xpert MTB/RIF-positive and RIF-resistant patients confirmed by LJ proportion tests,20 （76.9％） patients were infected by MDR-TB.Conclusions The Xpert MTB/RIF assay is a highly sensitive and specific method for diagnosis of TB and RIF resistance,which will enable it to have the potential to be used in county-level laboratories and lead to the reduction of the infectious pool and improvements in TB control in China.Further evaluations in county-level laboratories for implementing the assay are still required.展开更多
基金funded by the projects 2013ZX10003002-001 and 2013ZX10003006-002-001 of Chinese National Key Program of Mega Infectious Disease of the National 12th Five-Year Plan
文摘Objective A PCR-reverse dot blot hybridization （RDBH） assay was developed for rapid detection of rpoB gene mutations in ＇hot mutation region＇ of Mycobacterium tuberculosis （M. tuberculosis）. Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing （DST） and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2% （165/181） and 98.3% （117/119）, respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value （PPV） and negative predictive value （NPV） of the RDBH assay were 97.7% （293/300）, 98.2% （164/167）, and 97.0% （129/133）, respectively. Furthermore, the results indicated that the most common mutations were in codons 531 （48.6%）, 526 （25.4%）, 516 （8.8%）, and 511 （6.6%）, and the combinative mutation rate was 15 （8.3%）. One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.
基金This study was supported by a grant from the National Natural Science Foundation of China(No.30471841).
文摘Background China is one of the high burden countries of Mycobacterium tuberculosis （TB） infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin （RIF） and isoniazid （INH） resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance.Methods Forty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide （RIFO） assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA （-15C/T） and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism （RFLP） method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups. Results Sixty-five （91.5%） of 71 RIF resistant and 52 （92.9%） of 56 multidrug-resistant （MDR, i.e. resistant to at least RIF and INH） strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively, katG315 AGC〉ACC and inhA-15C〉T mutations were found in 40 （60.6%） and 10 （15.2%） of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into ＂modern＂ （76.6%） and ＂ancestral＂ （23.4%） group. There is no significant difference between ＂ancestral＂ and ＂modern＂ group in prevalance of drug resistance-associated gene mutations. Conclusions The hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains; a combined use of both katG315 and inhA-15 can improve the detection rate of I NH resistant strains; the Beijing genotype is prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.
文摘Background The Xpert MTB/RIF showed high sensitivity and specificity in previous studies carried out in different epidemiological and geographical settings and patient populations in high-burden tuberculosis （TB） countries.However,there were little data obtained by validation or demonstration study of the assay in China.In this study,the performance of Xpert MTB/RIF was investigated in two county-level laboratories in Hunan Province,China.Methods Consecutive patients with suspected pulmonary tuberculosis （PTB） and suspicion for multidrug-resistant tuberculosis （MDR-TB） were enrolled.For each patient suspected to have PTB,three sputum specimens （one spot sputum,one night sputum,and one morning sputum） were collected and each sputum was tested with smear microscopy,L（o）wenstein-Jensen （LJ） culture,and Xpert MTB/RIF test.For comparison across subgroups and testing methods,95％ confidence intervals were calculated.All analyses were done with SPSS 16.0,and P ＜0.05 was regarded as significant.Results For case detection,the sensitivity of Xpert MTB/RIF was 100％ for smear-and culture-positive TB and 88.6％ for smear-negative and culture-positive TB; the overall sensitivity was 94.5％ for all culture-positive patients.The specificity was 99.8％.The sensitivity of Xpert MTB/RIF assay was 22.0％ in clinical TB patients and the specificity reached 100.0％ in the group of patients who are infected with nontuberculous mycobacteria.For the detection of rifampin resistance,the sensitivity of MTB/RIF RIF-resistance detection was 92.9％,and the specificity was 98.7％.Of the 26 Xpert MTB/RIF-positive and RIF-resistant patients confirmed by LJ proportion tests,20 （76.9％） patients were infected by MDR-TB.Conclusions The Xpert MTB/RIF assay is a highly sensitive and specific method for diagnosis of TB and RIF resistance,which will enable it to have the potential to be used in county-level laboratories and lead to the reduction of the infectious pool and improvements in TB control in China.Further evaluations in county-level laboratories for implementing the assay are still required.
