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.

scholarly journals Detection of Mutations in 81-bpRifampin Resistance Determining Region (RRDR) of rpoB gene in Mycobacterium tuberculosis using GeneXpert MTB/RIF in Clinical Specimens from Quetta, Pakistan

2020 ◽  
Vol 53 (1) ◽  
Author(s):  
Muhammad Zahid Mengal ◽  
Hamida Ali ◽  
Raheela Asmat ◽  
Muhammad Naeem ◽  
Ferhat Abbas ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Fluorescent Microscopy ◽  
Simultaneous Detection ◽  
Surrogate Marker ◽  
Gene Mutations ◽  
Rpob Gene ◽  
Multidrug Resistant ◽  
Resistance Pattern ◽  
Clinical Specimens ◽  
Significant Difference

GeneXpert MTB/RIF has revolutionized the tuberculosis diagnosis by simultaneous detection of Mycobacterium tuberculosis and resistance to RIF (rifampicin), a surrogate marker for multidrug-resistant TB in less than two hours. The RIF-resistance pattern in Balochistan, Pakistan, is not documented. This study was aimed to detect RIF-resistant TB and mutations in RNA polymerase beta (rpoB) gene of M. tuberculosis within 81-bp RRDR in Quetta, Pakistan using GeneXpert® MTB/RIF assay. In total, 2300 clinical specimens were collected from suspected TB patients at Fatima Jinnah General and Chest Hospital Quetta, Pakistan between January and August 2017. These specimens were analyzed by GeneXpert® MTB/RIF assay. The data was statistically analyzed using SPSS software. Out of 2300 clinical specimens, M. tuberculosis was positive in in 899 (39.1%) cases by GeneXpert® MTB/RIF assay [positive respiratory cases 42.9% (871/2032) and non-respiratory 10.4% (28/268) with statistically significant difference (χ2= 104.5, p<0.001)]. Among 899 MTB positive cases, 46 (5.1%) were RIF-resistance caused by various rpoB gene mutations within 81-bp RRDR. Most of the RIF-resistant isolates were observed to harbor mutationsin Probe E 78.3% (n=36) whereas mutations in Probe A, B, D were observed 2.2% (n=1), 4.3% (n=2), and 6.5% (n=3), respectively. However, none of cases had RIF-resistance associated with Probe C. Out of 46 RRD cases, 21 (45.7 %) were males and 25 (54.3 %) were females. Additionally, Xpert® test showed higher detection rate than fluorescent microscopy (39.1% vs 31.2%, P<0.05) and detected MTB in 186 (11.8%) smear-negative specimens. Among 42 confirmed TB patients had MDR contact and eight patients were co-infected with HIV. In conclusion, 5.1% of the TB patients showed rifampicin resistance. The most frequent rpoB genetic mutations were observed in codons 531/533 (Probe E, 78.3%) whereas the least within the sequence 511 (Probe A, 2.2%).

scholarly journals Epidemiology of Rifampicin Resistant Tuberculosis and Common Mutations in rpoB Gene of Mycobacterium tuberculosis: A Retrospective Study from Six Districts of Punjab (India) Using Xpert MTB/RIF Assay

2016 ◽  
Vol 8 (02) ◽  
pp. 096-100 ◽  
Author(s):  
Ramandeep Kaur ◽  
Neerja Jindal ◽  
Shilpa Arora ◽  
Shajla Kataria
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Surrogate Marker ◽  
Rpob Gene ◽  
Multidrug Resistant ◽  
Resistance Pattern ◽  
Resistance Mutations ◽  
Resistant Tuberculosis ◽  
Mdr Tb ◽  
Previously Treated Patients ◽  
User Friendly

ABSTRACT Background: Xpert MTB/RIF assay has revolutionized the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (RIF), a surrogate marker for multidrug-resistant TB (MDR-TB) in <2 h. The RIF resistance pattern in Malwa region of Punjab, India, is not documented. Here, we report the epidemiology of RIF-resistant TB and mutations in rpoB gene of Mycobacterium tuberculosis (MTB). Materials and Methods: A total of 1612 specimens received between October 2013 and February 2015 were tested by Xpert MTB/RIF assay following manufacturer’s instructions. The results thus obtained were analyzed using SPSS version 20.0.0 (SPSS Inc., Chicago, IL, USA) statistical software. Result: RIF resistance was statistically higher in previously treated patients in comparison to the new patients (P = 0.006) and in patients with acid fast-Bacilli (AFB) positive smears to AFB-negative smears (P = 0.048). RIF resistance mutations in 130 specimens revealed frequency of E 73/130 (56%), B 28/130 (21.5%), D 18/130 (13.8%), A 11/130 (8.4%), and C 1/130 (0.7%) while in one specimen, mutation combination, i.e., mutations associated with more than one probe (A and B both) was present. Conclusion: Xpert MTB/RIF assay is a user-friendly screening tool for detection of MTB and RIF resistance from suspected TB/MDR cases in a shorter period of time. It could also serve as a useful technique to have simultaneous preliminary information regarding the mutation pattern of RIF resistance in MTB isolates.

scholarly journals Drug resistance rates of Mycobacterium tuberculosis strains in Austria between 1995 and 1998 and molecular typing of multidrug-resistant isolates

2000 ◽  
Vol 124 (3) ◽  
pp. 523-528 ◽  
Author(s):  
F. STAUFFER ◽  
A. MAKRISTATHIS ◽  
J. P. KLEIN ◽  
W. BAROUSCH ◽  
The Austrian Drug Resistant Tuberculosis Study Group
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Molecular Typing ◽  
Multidrug Resistant ◽  
Resistance Pattern ◽  
Drug Resistance Pattern ◽  
Resistant Strains ◽  
Resistance Rates

In this study the drug resistance pattern of 3559 Mycobacterium tuberculosis strains isolated in Austria between 1995 and 98 was evaluated. Of these strains, 165 (4·6%) were resistant to one or more drugs, 113 (3·2%) to one of the tested drugs and 53 (1·5%) to two or more drugs. Monodrug resistance was observed most often to isoniazid (56 strains), followed by streptomycin (44 strains). Resistance to rifampicin or ethambutol alone was rarely seen (12 strains and 1 strain, respectively). Of the 53 strains resistant to 2 or more drugs, 25 were resistant to isoniazid and streptomycin, while 17 were multidrug resistant. Molecular typing revealed a large diversity among the multidrug-resistant strains.

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.

scholarly journals Molecular Characterization of Drug-Resistant Mycobacterium tuberculosis Isolated from HIV-Positive Patients in Rivers State, Nigeria

2021 ◽  
pp. 44-51
Author(s):  
M. A. Alex-Wele ◽  
O. K. Obunge
Keyword(s):  
Gene Mutations ◽  
Multidrug Resistant ◽  
Resistance Pattern ◽  
Drug Resistant ◽  
Drug Resistance Pattern ◽  
Mdr Tb ◽  
Resistant Strains ◽  
Probe Assay ◽  
Gene 32 ◽  
Rivers State

Aim: Drug resistant tuberculosis is a major challenge in the global bid to control the disease burden and improve treatment outcomes of DRTB infected individuals. Methods: The Line Probe Assay (LPA) was used to assess the drug resistance pattern and gene mutations in 260 sputum specimens collected consecutively from 260 adult, HIV sero-positive subjects presenting with symptoms suggestive of TB in clinical settings across Rivers State, Nigeria. Results: The results showed a 61.2% (n = 159) prevalence of TB among all study subjects. LPA analysis showed that 16 (10.1%) were multidrug resistant strains, 17 (10.7%) Rifampicin (RIF) monoresistant strains, 24 (15.1%) Isonaizid (INH) monoresistant strains and 102 (64.2%) drug susceptible strains. Among all 32 RIF-resistant strains, 24 (75%) had a mutation in rpoB S531L at the MUT3 band. Another mutation was observed at rpoB H526D (1/17) in RIF-monoresistant strains but not in MDR-TB strains.  In INH resistant strains, mutations were observed at the katG gene [32/39 (82.1%)] which constituted 13/15 (86.7%) in MDR-TB strains and 19/24 (79.2%) in INH-monoresistant strains (p =0.002). Overall frequency of inhA mutation was 6/39 (15.4%); MDR-TB strains [2/15 (13.3%)] and INH-monoresistant strains [4/24 (16.7%), p = 0.2]. Combined KatG and inhA mutation was found in 1/39 (2.6%) of INH-monoresistant strains and in none of MDR-TB strains. Of the single inhA mutation bands, only 1/39 (4.2%) MUT 3B (T8A mutation) was observed in INH-monoresistant strains. Conclusion: The results showed that drug-resistant TB is prevalent in Rivers State with various gene mutations observed in the different TB strains isolated, making LPA analysis a necessity in the bid to improving prevention and control efforts in the region.

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