scholarly journals Mutations in the rpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Strains Isolated Mostly in Asian Countries and Their Rapid Detection by Line Probe Assay

1999 ◽  
Vol 37 (8) ◽  
pp. 2663-2666 ◽  
Author(s):  
Kazue Hirano ◽  
Chiyoji Abe ◽  
Mitsuyoshi Takahashi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rapid Detection ◽  
Rpob Gene ◽  
Line Probe Assay ◽  
Asian Countries ◽  
Nucleotide Substitutions ◽  
In Vitro Susceptibility ◽  
Nucleotide Insertion ◽  
Probe Assay

Mutations in the rpoB gene of 90 rifampin-resistantMycobacterium tuberculosis isolates mostly from Asian countries were analyzed. Ten distinct single-nucleotide substitutions were found among the isolates by automated sequencing. A 3-nucleotide insertion was found in two isolates, and no mutation was found in five isolates (5.6%). A reverse hybridization-based line probe assay (INNO-LiPA Rif TB) for rapid detection of the mutations was evaluated with these isolates. Concordance rates with sequencing results for five wild-type probes (S probes) and four probes for specific mutations (R probes) were 96.7 and 100%, respectively. The overall concordance rate with the in vitro susceptibility testing results was 92.2% (83 of 90 isolates). These results indicate that a commercial line probe assay kit may be useful for rapid diagnosis of rifampin-resistant tuberculosis.

scholarly journals Evaluation of a New Line Probe Assay for Rapid Identification of gyrA Mutations in Mycobacterium tuberculosis

2005 ◽  
Vol 49 (7) ◽  
pp. 2928-2933 ◽  
Author(s):  
Federico Giannoni ◽  
Elisabetta Iona ◽  
Federica Sementilli ◽  
Lara Brunori ◽  
Manuela Pardini ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rapid Identification ◽  
The Other ◽  
Nucleotide Sequencing ◽  
Line Probe Assay ◽  
Wild Type ◽  
In Vitro Susceptibility ◽  
Pcr Products ◽  
Probe Assay

ABSTRACT Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100% concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5%) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 × 103 M. tuberculosis CFU per ml of sputum.

scholarly journals MUTATIONS IN THE RPOB GENE OF MYCOBACTERIUM TUBERCULOSIS IDENTIFIED BY SEQUENCING METHOD

2017 ◽  
Vol 10 (3) ◽  
pp. 382
Author(s):  
Sureshbabu Ramalingam ◽  
Lakshmi Murali ◽  
Palaniswamy M
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tamil Nadu ◽  
Fluorescent Microscopy ◽  
Rpob Gene ◽  
Multidrug Resistant ◽  
Resistance Pattern ◽  
Line Probe Assay ◽  
Sequencing Method ◽  
Drug Resistance Pattern ◽  
Probe Assay

ABSTRACTObjective: To identify the mutation in the rpoB gene of Mycobacterium tuberculosis (MTB), using by sequencing method from pulmonary specimensof presumptive TB patients belonging to the districts of Tamil Nadu.Methods: A total of 8697 clinical specimens of presumptive MTB patients were collected from various districts of Tamil Nadu. Smear microscopy wasperformed by light emitting diode fluorescent microscopy and all the smear positive samples were tested using line probe assay (LPA) to detect thepercentage of drug resistance pattern and to identify the missing mutation in LPA by the sequencing method.Results: Among 4897 smear positives subjected to LPA method; 407 (8.3%) MTB was not detected and 16 (0.3%) showed invalid result; 4473 (91.4%)strains showed MTB positive; 3695 (82.6%) were sensitive for both rifampicin (RIF) and isoniazid (INH) drugs; 502 (11.2%) were resistance for INH;73 (1.6%) resistant for RIF; 203 (4.5%) were resistance for RIF and INH. Totally, 52 (1.2%) strains results cannot be confirmed by LPA and reportedas sensitive for RIF, because of the faint and the missing bands in both wild type and mutation. These strains were sequenced and 39 (75%) strainsshowed resistant to RIF.Conclusion: Hence LPA may be the molecular technology for the rapid, feasible and reliable method for the detection of multidrug resistant mutationbut few confusion bands cannot be reported as resistance, which should be confirmed by either conventional phenotypic drug susceptibility testingor by sequencing method.Keywords: Line probe assay, Sequencing, Mutation, Multidrug resistant tuberculosis.

AID TB resistance line probe assay for rapid detection of resistant Mycobacterium tuberculosis in clinical samples

2015 ◽  
Vol 70 (4) ◽  
pp. 400-408 ◽  
Author(s):  
B. Molina-Moya ◽  
A. Lacoma ◽  
C. Prat ◽  
J. Diaz ◽  
A. Dudnyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rapid Detection ◽  
Clinical Samples ◽  
Line Probe Assay ◽  
Probe Assay

scholarly journals Detection of mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains inhibiting wild type probe hybridization in the MTBDR plus assay by DNA sequencing directly from clinical specimens

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Pallavi Sinha ◽  
G. N. Srivastava ◽  
Rajneesh Tripathi ◽  
Mukti Nath Mishra ◽  
Shampa Anupurba
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Sequencing ◽  
Rpob Gene ◽  
Single Mutation ◽  
Line Probe Assay ◽  
Wild Type ◽  
Efficient Treatment ◽  
Clinical Specimens ◽  
Probe Hybridization ◽  
Probe Assay

Abstract Background The potential of genetic testing for rapid and accurate diagnosis of drug-resistant Mycobacterium tuberculosis strains is vital for efficient treatment and reduction in dissemination. MTBDR plus assays rapidly detect mutations related to drug resistance and wild type sequences allied with susceptibility. Although these methods are promising, the examination of molecular level performance is essential for improved assay result interpretation and continued diagnostic development. Therefore this study aimed to determine novel mutations that were inhibiting wild type probe hybridization in the Line probe assay by DNA sequencing. Using data collected from Line Probe assay (GenoType MTBDRplus assay) the contribution of absent wild type probe hybridization to the detection of rifampicin resistance was assessed via comparison to a reference standard method i.e. DNA sequencing. Results Sequence analysis of the rpoB gene of 47 MTB resistant strains from clinical specimens showed that 37 had a single mutation, 9 had double mutations and one had triple mutations in the ropB gene. Conclusions The absence of wild type probe hybridization without mutation probe hybridization was mainly the result of the failure of mutation probe hybridization and the result of the novel or rare mutations. Additional probes are necessary to be included in the Line probe assay to improve the detection of rifampicin-resistant Mycobacterium tuberculosis strains.

scholarly journals Evaluation of a line probe assay for the rapid detection of gyrA mutations associated with fluoroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis

2011 ◽  
Vol 60 (2) ◽  
pp. 184-188 ◽  
Author(s):  
Hiroki Ando ◽  
Satoshi Mitarai ◽  
Yuji Kondo ◽  
Toshinori Suetake ◽  
Seiya Kato ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Global Health ◽  
Dna Sequencing ◽  
Rapid Detection ◽  
Multidrug Resistant ◽  
Surveillance Study ◽  
Fluoroquinolone Resistance ◽  
National Surveillance ◽  
Line Probe Assay ◽  
Probe Assay

The aim of this study was to establish the importance of detecting fluoroquinolone (FQ) resistance in multidrug resistant (MDR) Mycobacterium tuberculosis, and to show the usefulness of a hybridization-based line probe assay (LiPA) for detecting gyrA mutations. Thirty-three MDR M. tuberculosis isolates were collected from a total of sixty MDR isolates identified in Japan over 6 months during a national surveillance study in 2002. Seventeen MDR isolates were collected by the National Center for Global Health and Medicine in Japan over 6 years from 2003 to 2008. These 50 isolates were examined for FQ susceptibility, and analysed by LiPA and gyrA sequencing. Among them, 22 (44 %) showed FQ resistance. All FQ-resistant isolates had at least one mutation in gyrA. The results of the LiPA were fully consistent with the DNA sequencing results. Given that on the basis of our results almost half of the MDR M. tuberculosis isolates in Japan might have resistance to FQ, it is important to monitor FQ resistance in patients with MDR tuberculosis (TB), as well as with drug-susceptible TB, prior to commencing treatment. For the detection of FQ resistance, LiPA is useful and can rapidly and efficiently assess FQ resistance.

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