scholarly journals Molecular characterization of rpoB gene mutations in isolates from tuberculosis patients in Cubal, Republic of Angola

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ariadna Rando-Segura ◽  
María Luisa Aznar ◽  
María Milagros Moreno ◽  
Mateu Espasa Soley ◽  
Elena Sulleiro Igual ◽  
...  
Keyword(s):  
Gene Mutations ◽  
Rpob Gene ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Resistance Pattern ◽  
Tuberculosis Patients ◽  
High Incidence ◽  
Resistant Strains

Abstract Background The importance of Mycobacterium tuberculosis strains with disputed rpoB mutations remains to be defined. This study aimed to assess the frequency and types of rpoB mutations in M. tuberculosis isolates from Cubal, Angola, a country with a high incidence of tuberculosis. Methods All isolates included (n = 308) were analyzed using phenotypic drug susceptibility testing and GenoType MTBDRplus assay. DNA sequencing of the rpoB gene and determination of rifampicin MIC by macrodilution method were additionally performed on isolates yielding discordant results (n = 12) and those in which the mutation detected was not characterized (n = 8). Results In total, 85.1% (74/87) of rifampicin-resistant strains had undisputed rpoB mutations -S450L (49), D435V (15), H445D (3), H445Y (2), Q432ins (1), L449M plus S450F (1), S450F (1), S450W (1) and S450Y (1)-; 10.3% (9/87) had disputed rpoB mutations—L430P plus S493L (1), N437del (1), H445L (3), D435Y (2), L452P (2)-, 2.3% (2.3%) showed no rpoB mutations and 2.3% (2/87) showed heteroresistance—D435Y plus L452P and L430P plus S493L-. Conclusion Disputed rpoB mutations were common, occurring in 10.3% of rifampicin resistant isolates. Current phenotyping techniques may be unable to detect this resistance pattern. To increase their sensitivity, a lower concentration of RIF could be used in these tests or alternatively, rpoB mutations could be screened and characterized in all M. tuberculosis strains.

scholarly journals Non-pncAGene-Mutated but Pyrazinamide-Resistant Mycobacterium tuberculosis: Why Is That?

2017 ◽  
Vol 55 (6) ◽  
pp. 1920-1927 ◽  
Author(s):  
Jim Werngren ◽  
Erik Alm ◽  
Mikael Mansjö
Keyword(s):  
Susceptibility Testing ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Molecular Techniques ◽  
Content Type ◽  
Resistant Strains ◽  
Clear Majority

ABSTRACTPyrazinamide (PZA) is a key component for the effective treatment of drug-susceptible and PZA-susceptible multidrug-resistant (MDRPZA-S) tuberculosis (TB).pncAgene mutations are usually detected in a clear majority (>90%) of PZA-resistant strains but obviously not in all. Rapid and reliable PZA drug susceptibility testing (DST) is critical whenever PZA is to be used in a treatment regimen, not least for the treatment of MDRPZA-STB. In this study, we selected 26 PZA-resistant isolates reported to carry a wild-typepncAgene. To confirm resistance, susceptibility testing was repeated using 100 mg/liter and 200 mg/liter PZA for all the 26 isolates and Sanger sequencing was repeated on the 18 isolates that remained PZA resistant. Apart from the eight isolates initially misclassified as PZA resistant, the retests identified three factors responsible for the phenotype-genotype discrepancy:panDorrpsAmutations identified by whole-genome sequencing (WGS) (n= 7), heteroresistance (n= 8), and mixed populations withMycobacterium avium(n= 3). Additionally, we performed WGS on 400 PZA-susceptible isolates and 15 consecutive MDRPZA-Rclinical isolates. Of the 400 PZA-susceptible isolates, only 1 harbored a nonsynonymouspncAmutation (Thr87Met), whereas a nonsynonymousrpsAmutation was found in 17 isolates. None of these isolates carried a nonsynonymouspanDmutation, while all 15 of the MDRPZA-Risolates harbored a nonsynonymouspncAmutation. Our findings indicate that it is necessary to consider the occurrence ofpanDmutations in PZA-resistant isolates, as well as heteroresistance, for the development and evaluation of new molecular techniques to ensure high-quality DST performance. The identification of nonsynonymousrpsAmutations in both PZA-susceptible and PZA-resistant isolates also implies that further studies are needed in order to determine the role ofrpsAin PZA resistance.

scholarly journals rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China

2021 ◽  
Vol 49 (3) ◽  
pp. 030006052199759
Author(s):  
Mei-Chun Zeng ◽  
Qing-Jun Jia ◽  
Lei-Ming Tang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rural Areas ◽  
Gene Mutations ◽  
Gene Sequencing ◽  
Rpob Gene ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Drug Resistant ◽  
Extensively Drug Resistant

Objective The aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China. Methods We prospectively analyzed RIFR-associated mutations in 13 rural areas of Zhejiang. Isolates were subjected to species identification, phenotype drug susceptibility testing (DST), DNA extraction, and rpoB gene sequencing. Results A total of 103 RIFR isolates were identified by DST (22 RIFR only, 14 poly-drug resistant, 49 multidrug resistant, 13 pre-extensively drug resistant [pre-XDR], and 5 extensively drug resistant [XDR]) from 2152 culture-positive sputum specimens. Gene sequencing of rpoB showed that the most frequent mutation was S450L (37.86%, 39/103); mutations P280L, E521K, and D595Y were outside the rifampicin resistance-determining region (RRDR) but may be associated with RIFR. Mutations associated with poly-drug resistant, pre-XDR, and XDR TB were mainly located at codon 445 or 450 in the RRDR. Conclusions The frequency of rpoB RRDR mutation in Zhejiang is high. Further studies are needed to clarify the relationships between RIFR and the TTC insertion at codon 433 in the RRDR and the P280L and D595Y mutations outside the RRDR.

Molecular characterization of rpoB gene mutations in rifampicine-resistantMycobacterium tuberculosis isolates from tuberculosis patients in Belarus

2006 ◽  
Vol 1 (12) ◽  
pp. 1447-1452 ◽  
Author(s):  
Leonid P. Titov ◽  
Saeed Zakerbostanabad ◽  
Veranika Slizen ◽  
Larisa Surkova ◽  
Mohammad Taghikhani ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Gene Mutations ◽  
Rpob Gene ◽  
Tuberculosis Patients

scholarly journals Reevaluation of the Critical Concentration for Drug Susceptibility Testing of Mycobacterium tuberculosis against Pyrazinamide Using Wild-Type MIC Distributions andpncAGene Sequencing

2011 ◽  
Vol 56 (3) ◽  
pp. 1253-1257 ◽  
Author(s):  
J. Werngren ◽  
E. Sturegård ◽  
P. Juréen ◽  
K. Ängeby ◽  
S. Hoffner ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Clinical Isolates ◽  
Wild Type ◽  
First Line ◽  
Content Type ◽  
Resistant Strains

ABSTRACTPyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistantMycobacterium tuberculosisstrains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates ofMycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinicalM. tuberculosisisolates and compared the results topncAsequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n= 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤8 to 64 mg/liter), and nopncAgene mutations were detected. In strains resistant in both Bactec systems (n= 18), the PZA MICs ranged from 256 to ≥1,024 mg/liter. The discordances betweenpncAsequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated topncAmutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

scholarly journals Whole Genome Sequencing for the Analysis of Drug Resistant Strains of Mycobacterium tuberculosis: A Systematic Review for Bedaquiline and Delamanid

Antibiotics ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 133
Author(s):  
Luisa Maria Nieto Ramirez ◽  
Karina Quintero Vargas ◽  
Gustavo Diaz
Keyword(s):  
Systematic Review ◽  
Gene Mutations ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Drug Resistant ◽  
Drug Regimens ◽  
Resistant Strains ◽  
One Year ◽  
Drug Resistant Strains ◽  
Different Levels

Tuberculosis (TB) remains the deadliest Infectious disease worldwide, partially due to the increasing dissemination of multidrug and extensively drug-resistant (MDR/XDR) strains. Drug regimens containing the new anti-TB drugs bedaquiline (BDQ) and delamanid (DLM) appear as a last resort for the treatment of MDR or XDR-TB. Unfortunately, resistant cases to these drugs emerged just one year after their introduction in clinical practice. Early detection of resistant strains to BDQ and DLM is crucial to preserving the effectiveness of these drugs. Here, we present a systematic review aiming to define all available genotypic variants linked to different levels of resistance to BDQ and DLM that have been described through whole genomic sequencing (WGS) and the available drug susceptibility testing methods. During the review, we performed a thorough analysis of 18 articles. BDQ resistance was associated with genetic variants in Rv0678 and atpE, while mutations in pepQ were linked to a low-level of resistance for BDQ. For DLM, mutations in the genes ddn, fgd1, fbiA, and fbiC were found in phenotypically resistant cases, while all the mutations in fbiB were reported only in DLM-susceptible strains. Additionally, WGS analysis allowed the detection of heteroresistance to both drugs. In conclusion, we present a comprehensive panel of gene mutations linked to different levels of drug resistance to BDQ and DLM.

Accuracy of molecular drug susceptibility testing amongst tuberculosis patients in Karakalpakstan, Uzbekistan

2021 ◽  
Author(s):  
Horacio Gil ◽  
Hasmik Margaryan ◽  
Ismailov Azamat ◽  
Bekturdieva Ziba ◽  
Halmuratov Bayram ◽  
...  
Keyword(s):  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Tuberculosis Patients

scholarly journals How Well Do Routine Molecular Diagnostics Detect Rifampin Heteroresistance in Mycobacterium tuberculosis?

2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Kamela C. S. Ng ◽  
Philip Supply ◽  
Frank G. J. Cobelens ◽  
Cyril Gaudin ◽  
Julian Gonzalez-Martin ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Drug Susceptibility Testing ◽  
Tb Treatment ◽  
Proportion Method ◽  
Targeted Deep Sequencing ◽  
Resistant Strains ◽  
Rifampin Resistance ◽  
To Receive

ABSTRACT Rifampin heteroresistance—where rifampin-resistant and -susceptible tuberculosis (TB) bacilli coexist—may result in failed standard TB treatment and potential spread of rifampin-resistant strains. The detection of rifampin heteroresistance in routine rapid diagnostic tests (RDTs) allows for patients to receive prompt and effective multidrug-resistant-TB treatment and may improve rifampin-resistant TB control. The limit of detection (LOD) of rifampin heteroresistance for phenotypic drug susceptibility testing by the proportion method is 1% and, yet, is insufficiently documented for RDTs. We, therefore, aimed to determine, for the four RDTs (XpertMTB/RIF, XpertMTB/RIF Ultra, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII), the LOD per probe and mutation, validated by CFU counting and targeted deep sequencing (Deeplex-MycTB). We selected one rifampin-susceptible and four rifampin-resistant strains, with mutations D435V, H445D, H445Y, and S450L, respectively, mixed them in various proportions in triplicate, tested them with each RDT, and determined the LODs per mutation type. Deeplex-MycTB revealed concordant proportions of the minority resistant variants in the mixtures. The Deeplex-MycTB-validated LODs ranged from 20% to 80% for XpertMTB/RIF, 20% to 70% for Xpert Ultra, 5% to 10% for GenoTypeMTBDRplusv2.0, and 1% to 10% for GenoscholarNTM+MDRTBII for the different mutations. Deeplex-MycTB, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII provide explicit information on rifampin heteroresistance for the most frequently detected mutations. Classic Xpert and Ultra report rifampin heteroresistance as rifampin resistance, while Ultra may denote rifampin heteroresistance through “mixed patterns” of wild-type and mutant melt probe, melt peak temperatures. Overall, our findings inform end users that the threshold for reporting resistance in the case of rifampin heteroresistance is the highest for Classic Xpert and Ultra to resolve phenotypic and genotypic discordant rifampin-resistant TB results.

PREVALANCE OF GENETIC MUTATION ASSOSIATED WITH (RPOB) RIFAMPICIN RESISTANT AMONG THE TREATED TUBERCULOSIS PATIENTS

2020 ◽  
Vol 11 (12) ◽  
pp. 235-268
Author(s):  
Girija Vasudevan ◽  
Muthuraj M ◽  
Antony V samrot
Keyword(s):  
Drug Susceptibility ◽  
Genetic Mutation ◽  
Drug Susceptibility Testing ◽  
Nucleic Acid Amplification ◽  
Drug Resistant ◽  
Tuberculosis Patients ◽  
Resistant Tuberculosis ◽  
Resource Limited ◽  
Probe Assay ◽  
Solid Liquid

Tuberculosis (TB) is an infectious disease caused predominantly by Mycobacterium tuberculosis. India accounts for one fourth of the global TB burden. Drug-resistant tuberculosis (TB) undermines control efforts and its burden is poorly understood in resource-limited settings. Drug resistant TB is a laboratory based diagnosis and is performed either by phenotypic drug susceptibility testing using solid/liquid culture or genotypic testing for detection of resistance by Line Probe Assay/Cartridge based Nucleic Acid Amplification tests.

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