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1.
Mutations prevalent among rifampin- and isoniazid-resistant Mycobacterium tuberculosis isolates from a hospital in Vietnam
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Vietnam is ranked 13th among the WHO list of 22 high-burden countries, based upon estimated total number of tuberculosis cases. Despite having a model national tuberculosis program, consistently achieving and exceeding WHO targets for detection and cure, drug-resistant and multidrug-resistant tuberculosis cases continue to rise. Rapid multidrug-resistant tests applicable in this setting, coupled with effective treatment regimens, would be a useful tool in reversing this trend, allowing early identification of patients with multidrug-resistant tuberculosis and avoiding resistance-amplifying regimens. Sequencing of consecutive isolates identified by the National Tuberculosis Program showed 89% of isoniazid-resistant isolates could be detected by targeting just 2 codons, katG 315 and -15C-->T in the inhA promoter, while rifampin resistance will be more complex to detect, with many different mutation and insertion events in rpoB. The most prevalent rifampin resistance-conferring mutations, as in other countries, were in rpoB codons 531 (43%), 526 (31%), and 516 (15%). However, a hybridization-based resistance test with probes targeting the 5 most common mutations would only detect 78% of rifampin-resistant isolates. Overall, these data suggest that rifampin resistance may be used as a surrogate marker for multidrug-resistant tuberculosis and that a sensitivity of between 70 to 80% may be possible for rapid molecular detection of multidrug-resistant tuberculosis in this setting. 相似文献
2.
《Indian journal of medical microbiology》2014,32(4):398-403
Background: Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. Materials and Methods: High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings. 相似文献
3.
The worldwide threat of drug-resistant tuberculosis (TB) to human health has led to the development of molecular methods for rapidly determining the resistance of clinical Mycobacterium tuberculosis isolates to the two front-line antituberculous drugs, isoniazid and rifampin. The prevalence of the S315T mutation within the katG gene, which confers clinically significant resistance to isoniazid, was determined in isoniazid-resistant clinical M. tuberculosis isolates recovered from TB patients in Kuwait. A total of 67 isoniazid-resistant and 18 susceptible clinical M. tuberculosis isolates were tested. The mutation S315T was found in 46 (69%) of the 67 resistant strains, whereas none of the susceptible strains contained this mutation. The prevalence of this mutation was highest (32 of 40, 80%) in isolates recovered from patients of South Asian origin and lowest in isolates from patients of Middle Eastern origin (8 of 18, 44%). The genotyping performed on isolates carrying the S315T mutation showed that the isolates belong to several different types as they exhibited unique DNA banding patterns. The results point to a varying prevalence of the S315T mutation within the katG gene in clinical M. tuberculosis isolates recovered from patients of different ethnic groupings within the same country. The results also suggest that detection of the S315T mutation in the katG gene may be used as a rapid screening method for identifying isoniazid-resistant clinical M. tuberculosis isolates recovered from majority of patients in some ethnic groupings. 相似文献
4.
Azar Dokht Khosravi Hossein Meghdadi Ata A. Ghadiri Ameneh Alami Amir Hossein Sina Mehdi Mirsaeidi 《APMIS : acta pathologica, microbiologica, et immunologica Scandinavica》2018,126(3):241-247
The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin‐fixed, paraffin‐embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty‐seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin‐resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates. 相似文献
5.
C. Yao T. Zhu Y. Li L. Zhang B. Zhang J. Huang W. Fu 《Clinical microbiology and infection》2010,16(11):1639-1643
The emergence of multidrug-resistance Mycobacterium tuberculosis is an increasing threat to tuberculosis control programmes. Susceptibility testing of Mycobacterium tuberculosis complex isolates by traditional methods requires a minimum of 14 days. This can be reduced significantly if molecular analysis is used. DNA sequencing is a good method for detecting mutation, but cannot be used routinely because of its relatively high cost. A sensitive and specific microarray has been designed to detect mutations in the rifampin resistance determining region of rpoB and loci in katG and inhA associated with isoniazid (INH) resistance. A panel of Mycobacterium tuberculosis isolates containing 13 different rpoB genotypes, two mutation genotypes within codon 315 of katG and one mutation genotypes at inhA was used to validate the microarray. The results obtained indicate that 100% of rifampicin-resistant M. tuberculosis strains isolated in Chongqing had rpoB mutations, with 531-Ser and 526-His being the most common positions substituted. Of the total 50 INH resistant isolates, 82% had a katG315 mutation and 18% had an inhA mutation. All the mutations detected by the microarray method were also confirmed by conventional DNA sequencing. It is demonstrated that the microarray is an efficient, specialized technique and can be used as a rapid method for detecting rifampin and isoniazid resistance. 相似文献
6.
Molecular characterization of rifampin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Poland 总被引:7,自引:0,他引:7
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Sajduda A Brzostek A Poplawska M Augustynowicz-Kopec E Zwolska Z Niemann S Dziadek J Hillemann D 《Journal of clinical microbiology》2004,42(6):2425-2431
A total of 105 rifampin (RMP)- and/or isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different parts of Poland in 2000 were screened for mutations associated with resistance to these drugs by two molecular methods, namely sequence analysis and real-time PCR technology. Three loci associated with drug resistance were selected for characterization: they were rpoB (RMP), katG, and the regulatory region of inhA (INH). Nineteen different mutations were identified in 64 RMP-resistant strains, and five new alleles were described. The most common point mutations were in codons 531 (41%), 516 (16%), and 526 (9%) of the rpoB gene. Mutations were not found in two (3%) of the isolates. In the case of resistance to INH, six different mutations in the katG gene of 83 resistant strains were detected. Fifty-seven (69%) isolates exhibited nucleotide substitutions at codon 315. One strain harbored a mutation affecting codon 279 (Gly279Thr). Twelve of 26 INH-resistant strains with the wild-type codon 315 (14.5% of all strains tested) had the mutation -15C-->T in the regulatory region of inhA. A full correlation between the DNA sequence analysis and real-time PCR data was obtained. We conclude that the real-time PCR method is fast and reliable for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates. 相似文献
7.
Karahan ZC Atalay F Uzun M Erturan Z Atasever M Akar N 《Microbial drug resistance (Larchmont, N.Y.)》2004,10(4):325-333
Drug-resistant tuberculosis is a serious problem throughout the world. Resistance to Rifampicin (RIF) is mainly caused by the mutations in the rpoB gene coding the beta-subunit of RNA polymerase. In this study, we aimed to detect the distribution of rpoB gene mutations in 80 RIF-resistant clinical Mycobacterium tuberculosis (MTB) isolates from Turkey. The rpoB gene was amplified by PCR and mutations leading to RIF resistance were determined by automated sequence analysis. A total of 72 of the 80 isolates (90%) were found to carry mutations in the amplified region, whereas eight isolates (10%) carried no mutations. Overall, 24 different missense mutations affecting 14 codons, and two deletion mutants were identified. Nine new mutations, six in the hot-spot region and three outside this region, were found. The codon numbers of the most frequently encountered mutations were 531 (51.4%), 526 (18.1%), 516 (13.9%), and 513 (12.5%). As a result, 90% of the RIF-resistant MTB isolates from the Turkish patients were found to carry a mutation in the rpoB gene, Ser531Leu being the most frequent one. Although molecular methods identify mutations leading to RIF resistance very quickly, results of the antimycobacterial susceptibility tests must be taken into consideration for the patients carrying no mutations in this region. 相似文献
8.
Mutations in katG,inhA, and ahpC genes of Brazilian isoniazid-resistant isolates of Mycobacterium tuberculosis 总被引:2,自引:0,他引:2
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Silva MS Senna SG Ribeiro MO Valim AR Telles MA Kritski A Morlock GP Cooksey RC Zaha A Rossetti ML 《Journal of clinical microbiology》2003,41(9):4471-4474
The presence of mutations in specific regions of the katG, inhA, and ahpC genes was analyzed with 69 Mycobacterium tuberculosis isoniazid-resistant isolates from three Brazilian states. Point mutations in codon 315 of the katG gene were observed in 87.1, 60.9, and 60% of the isolates from Rio Grande do Sul, Rio de Janeiro, and S?o Paulo, respectively. Mutations in the inhA gene were identified only in one isolate from RJ State, and the ahpC promoter region revealed mutations in distinct positions in 12.9, 21.7, and 6.7% of the isolates from RS, RJ and SP, respectively. 相似文献
9.
Detection of isoniazid-resistant Mycobacterium tuberculosis strains by a multiplex allele-specific PCR assay targeting katG codon 315 variation 总被引:5,自引:0,他引:5
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Mokrousov I Otten T Filipenko M Vyazovaya A Chrapov E Limeschenko E Steklova L Vyshnevskiy B Narvskaya O 《Journal of clinical microbiology》2002,40(7):2509-2512
We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC-->ACC and ACA (Ser-->Thr) substitutions that confer resistance to isoniazid (INH) in Mycobacterium tuberculosis clinical isolates. The 315 ACC allele is found in the majority of Inh(r) strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States. The mutations in katG315 are revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. Initially optimized on the purified DNA samples, the assay was then tested on crude cell lysates and auramine-stained sputum slide preparations with the same reproducibility and interpretability of profiles generated by agarose gel electrophoresis. The MAS-PCR assay can be used for the detection of resistance to INH in clinical laboratories in regions with a high prevalence of MDR M. tuberculosis strains. 相似文献
10.
Rapid genotypic detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis directly in clinical specimens
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A multiplex PCR DNA strip assay (Genotype MTBDR) designed to detect rifampin (rpoB) and high-level isoniazid (katG) resistance mutations in Mycobacterium tuberculosis isolates was optimized for clinical specimens. Successful genotypic results were achieved with 36 of 38 (95%) smear-positive respiratory specimens, allowing rapid therapeutic adjustments in transmittable drug-resistant tuberculosis. 相似文献
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13.
Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical Mycobacterium tuberculosis isolates 总被引:5,自引:0,他引:5
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Heep M Brandstätter B Rieger U Lehn N Richter E Rüsch-Gerdes S Niemann S 《Journal of clinical microbiology》2001,39(1):107-110
The prevalence of recently described mutation V176F, located in the beginning of the rpoB gene and associated with rifampin resistance and the wild-type cluster I sequence, was determined by analyzing the distribution of rpoB mutations among 80 rifampin (RIF)-resistant Mycobacterium tuberculosis strains isolated in Germany during 1997. The most frequent rpoB mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%) and codon 451 (11 isolates, 14%). The V176F mutation was detected in one isolate of the study population and in 5 of 18 RIF-resistant strains with no cluster I mutation from six previously published studies. In three isolates, a mixture of resistant and susceptible subpopulations (heteroresistance) prohibited the detection of rpoB mutations in the initial analysis; however, in these isolates, cluster I mutations could be verified after a passage on RIF-containing medium. IS6110 DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical restriction fragment length polymorphism patterns that mainly also show identical rpoB mutations and identical or similar drug resistance patterns. In conclusion, our results indicate that the V176F mutation should be included in molecular tests for prediction of RIF resistance in M. tuberculosis. We further demonstrated that heteroresistance caused by a mixture of mycobacterial subpopulations with different susceptibilities to RIF may influence the sensitivity of molecular tests for detection of resistance. 相似文献
14.
M. Y. Lipin V. N. Stepanshina I. G. Shemyakin T. M. Shinnick 《Clinical microbiology and infection》2007,13(6):620-626
Most multidrug-resistant (MDR) Mycobacterium tuberculosis isolates in Russia belong to the Beijing or Latino-American and Mediterranean (LAM) spoligotype families. The objective of this study was to investigate possible associations between genotype and the frequencies of mutations that confer drug resistance in a population that has two large families of circulating strains. Spoligotyping, IS6110 restriction fragment length polymorphism typing, and sequencing of the katG and rpoB genes, were performed for 217 consecutive MDR M. tuberculosis isolates from patients. The rpsL and rrs genes were also sequenced for selected streptomycin-resistant isolates. Of the 217 MDR isolates, 99 (46%) belonged to the LAM family, 92 (42%) to the Beijing family, 21 (10%) to the Haarlem family and four (2%) to the T family. There was one unique spoligotype. Mutations in the katG gene were identified in 207 (95%) isolates, all of which had mutations in codon 315. Mutations in the rpoB gene were identified in 200 (92%) isolates; 75% of LAM isolates carried a mutation in codon 516, whereas 71% of Beijing isolates carried a mutation in codon 531. In the 33 isolates resistant to streptomycin 50 mg/L, the 43AGG rpsL mutation was found in 27% of Haarlem, 75% of Beijing and 0% of LAM isolates, and rrs mutations were found in 17% (516C-->T) of Beijing and 100% (513A-->C) of LAM isolates. Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampicin or streptomycin in the Beijing and LAM families. The biological implications of this correlation remain to be explored. 相似文献
15.
Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR
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Ruiz M Torres MJ Llanos AC Arroyo A Palomares JC Aznar J 《Journal of clinical microbiology》2004,42(4):1585-1589
Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (> or = 0.1 microg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens. 相似文献
16.
Bostanabad SZ Titov LP Bahrmand A Nojoumi SA 《Indian journal of medical microbiology》2008,26(2):143-147
The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Forty two isoniazid-resistant isolates were identified from sputum of 163 patients with active pulmonary tuberculosis. Drug susceptibility testing was determined by using CDC standard conventional proportional method and BACTEC system. Standard PCR method for detection of isoniazid resistance associated mutations was performed by katG gene amplification and DNA sequencing. Most mutations were found in katG gene codons 315, 316 and 309. Four types of mutations were identified in codon 315: AGC-->ACC (n=36) 85%, AGC-->AGG (n=1) 2.3%, AGC-->AAC (n=2) 4.7%, AGC-->GGC (n=1) 2.3%. One type of mutation was found in codon 316: GGC-->AGC (n=18) 41.4%, four types of mutations were detected in codon 309: GGT-->GGT (n=7) 16.1%, GGT-->GCT (n=4) 9.2%, GGT-->GTC (n=3)6.9%, GGT-->GGG (n=1) 2.7%. The highest frequency of mutations sharing between primary and secondary infections was found in codon 315. 相似文献
17.
In-vitro activity of rifabutin against rifampicin-resistant Mycobacterium tuberculosis isolates with known rpoB mutations 总被引:2,自引:0,他引:2
The relationship between resistance to rifampicin and rifabutin and genetic alterations in the rpoB gene of 41 rifampicin-resistant isolates of Mycobacterium tuberculosis was evaluated. Although 35 isolates with rifampicin MICs > or = 32 mg/L were also rifabutin-resistant, six isolates with rifampicin MICs of 2-16 mg/L were susceptible to rifabutin (MIC < or = 0.5 mg/L). Mutations Asp516Val, Asp516Tyr, Leu533Pro and the double mutation Met515Ile and Leu533Pro influenced susceptibility to rifampicin, but not to rifabutin. All mutations at codons 531 and 526, except one isolate with a His526Cys mutation, correlated with resistance to both compounds. 相似文献
18.
rpoB Gene mutations and molecular characterization of rifampin-resistant Mycobacterium tuberculosis isolates from Shandong Province, China 总被引:4,自引:0,他引:4
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Ma X Wang H Deng Y Liu Z Xu Y Pan X Musser JM Graviss EA 《Journal of clinical microbiology》2006,44(9):3409-3412
Sixty rifampin (RIF)-resistant and 75 RIF-susceptible Mycobacterium tuberculosis isolates from Shandong Province, China, were analyzed for rpoB gene mutations and genotyped. Mycobacterial interspersed repetitive unit (MIRU) genotype 223325173533 was overrepresented among RIF-resistant isolates. MIRU combined with IS6110 restriction fragment length polymorphism analysis as the second-line genotyping method may reflect epidemiologic links more reliably than each method alone. 相似文献
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Zhou A Nawaz M Duan Y Moore JE Millar BC Xu J Yao Y 《Microbial drug resistance (Larchmont, N.Y.)》2011,17(2):275-281
The increased prevalence of isoniazid (INH)-resistant tuberculosis (TB) has become a significant challenge for TB control in the last 10 years. It has been reported that INH resistance is most frequently associated with katG 315 and inhA-15 mutations. The relative significance of these mutations in INH-resistant Mycobacterium tuberculosis and their association with the different genotypes of M. tuberculosis in Xi'an, China, was investigated. Eighty-nine INH-resistant isolates were subjected to molecular characterization of hotspot mutations within katG 315 and inhA-15 by Multiplex allele-specific PCR, and results from 10 isolates were confirmed by direct sequencing. Mutations in katG 315 and inhA-15 were detected in 58 and 7 isolates, respectively. These isolates were genotyped by mycobacterial interspersed repetitive unit-variable number of tandem repeats and Spoligotyping. Spoligotyping showed that 79 isolates (89%) belonged to Beijing family. Mycobacterial interspersed repetitive unit-variable number of tandem repeats typing revealed significant differences in clustering level between Beijing and non-Beijing family (56% vs. 9%, p?=?0.002), which indicated the active transmission of Beijing family in INH-resistant isolates. Our results suggested that the katG 315 mutants were predominant in INH-resistant isolates in Xi'an and the hotspot mutations within katG 315 and inhA-15 can be used as genetic markers for detection of INH resistance. 相似文献