scholarly journals Discordance between Xpert MTB/RIF Assay and Bactec MGIT 960 Culture System for Detection of Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Country with a Low Tuberculosis (TB) Incidence

2015 ◽  
Vol 53 (4) ◽  
pp. 1351-1354 ◽  
Author(s):  
Eiman Mokaddas ◽  
Suhail Ahmad ◽  
Hanaa S. Eldeen ◽  
Noura Al-Mutairi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Culture System ◽  
Content Type ◽  
Rifampin Resistance ◽  
Xpert Assay

Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10Mycobacterium tuberculosissamples were detected as rifampin susceptible and rifampin resistant, respectively. Two isolates that were rifampin susceptible by the MGIT system were rifampin resistant by the Xpert assay.rpoBsequencing identified a silent (CTG521TTG) mutation in one isolate and a missense (GAC516TAC) mutation in another. The detection of rifampin resistance is imperfect with both the Xpert assay and MGIT system. Any discordant rifampin resistance results should be confirmed by sequencing of therpoBgene.

scholarly journals Retrospective Analysis of False-Positive and Disputed Rifampin Resistance Xpert MTB/RIF Assay Results in Clinical Samples from a Referral Hospital in Hunan, China

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Peilei Hu ◽  
Hongtai Zhang ◽  
Joy Fleming ◽  
Guofeng Zhu ◽  
Shuai Zhang ◽  
...  
Keyword(s):  
Treatment Outcomes ◽  
False Positive ◽  
Culture System ◽  
Clinical Samples ◽  
Content Type ◽  
Previously Treated Patients ◽  
Previously Treated ◽  
Rifampin Resistance ◽  
Mgit Culture ◽  
Xpert Assay

ABSTRACT Concerns about the specificity of the Xpert MTB/RIF (Xpert) assay have arisen, as false-positive errors in the determination of Mycobacterium tuberculosis complex (MTBC) infection and rifampin (RIF) resistance in clinical practice have been reported. Here, we investigated 33 cases where patients were determined to be RIF susceptible using the Bactec MGIT 960 (MGIT) culture system but RIF resistant using the Xpert assay. Isolates from two of these patients were found not to have any mutations in the rifampin resistance determining region (RRDR) region of rpoB and had good treatment outcomes with first-line antituberculosis (anti-TB) drugs. The remaining 31 patients included 5 new cases and 26 previously treated patients. A large number of well-documented disputed mutations, including Leu511Pro, Asp516Tyr, His526Asn, His526Leu, His526Cys, and Leu533Pro, were detected, and mutations, including a 508 to 509 deletion and His526Gly, were described here as disputed mutations for the first time. Twenty-one (81%) of the 26 previously treated patients had poor treatment outcomes, and isolates from 19 (90%) of these 21 patients were resistant to isoniazid (INH) as determined using the MGIT culture system. Twenty-seven of the 31 isolates with disputed rpoB mutations were phenotypically resistant to INH, 21 (78%) being predicted by GenoType MTBDRplus to have a high level of INH resistance. Most (77.4%) of the isolates with disputed mutations were of the Beijing lineage. These findings have implications for the interpretation of false-positive and disputed rifampin resistance Xpert MTB/RIF results in clinical samples and provide guidance on how clinicians should manage patients carrying isolates with disputed rpoB mutations.

scholarly journals Molecular Analysis of Codon 548 in therpoBGene Involved in Mycobacterium tuberculosis Resistance to Rifampin

2014 ◽  
Vol 59 (3) ◽  
pp. 1542-1548 ◽  
Author(s):  
Yu-Tze Horng ◽  
Wen-Yih Jeng ◽  
Yih-Yuan Chen ◽  
Che-Hung Liu ◽  
Horng-Yunn Dou ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Resistance ◽  
Drug Susceptibility ◽  
Content Type ◽  
Stable Hydrogen Bond ◽  
Resistance Patterns ◽  
Resistant Strains ◽  
Rifampin Resistance ◽  
Susceptibility Tests ◽  
Dna Complex

ABSTRACTMostMycobacterium tuberculosisrifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the generpoB. However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates contained mutations both within and outside the RRDR, and only one isolate had a single missense mutation (Arg548His) located outside the RRDR. A drug susceptibility test of recombinantMycobacterium smegmatisandM. tuberculosisisolates carrying mutatedrpoB(Arg548His) showed an increased MIC for rifampin compared to that of the control strains. Modeling of the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain formed a more stable hydrogen bond structure than did Arg548, reducing the flexibility of the rifampin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant does not effectively interact with rifampin and results in bacterial resistance to the drug. This is the first report on the relationship between the mutation in codon 548 of RpoB and rifampin resistance in tuberculosis. The novel mutational profile of therpoBgene described here will contribute to the comprehensive understanding of rifampin resistance patterns and to the development of a useful tool for simple and rapid drug susceptibility tests.

scholarly journals Acquisition of Rifampin Resistance in Pulmonary Tuberculosis

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Xavier A. Kayigire ◽  
Sven O. Friedrich ◽  
Lize van der Merwe ◽  
Andreas H. Diacon
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pulmonary Tuberculosis ◽  
Drug Distribution ◽  
Newly Diagnosed ◽  
Spontaneous Mutations ◽  
Content Type ◽  
Tuberculosis Patients ◽  
Fixed Drug ◽  
Rifampin Resistance ◽  
Temporal And Spatial

ABSTRACT Mycobacterium tuberculosis strains with spontaneous mutations conferring resistance to rifampin (RIF) are exceedingly rare, and fixed drug combinations typically prevent augmentation of resistance to single drugs. Fourteen newly diagnosed tuberculosis patients were treated with RIF alone for 14 days, and bacterial loads, including mutation frequencies, were determined. A statistical model estimated that 1% of the remaining viable mycobacteria could be RIF resistant after 30 days of monotherapy. This indicates that temporal and spatial windows of RIF monotherapy due to uneven drug distribution within lung lesions could contribute to the acquisition of resistance to RIF.

scholarly journals Genetic Mimetics of Mycobacterium tuberculosis and Methicillin-Resistant Staphylococcus aureus as Verification Standards for Molecular Diagnostics

2017 ◽  
Vol 55 (12) ◽  
pp. 3384-3394 ◽  
Author(s):  
Edith Erika Machowski ◽  
Bavesh Davandra Kana
Keyword(s):  
Staphylococcus Aureus ◽  
Mycobacterium Tuberculosis ◽  
Molecular Diagnostics ◽  
Nucleic Acid Amplification ◽  
World Health ◽  
Molecular Diagnostic ◽  
Specific Gene ◽  
Methicillin Resistant ◽  
Content Type ◽  
Rifampin Resistance

ABSTRACTMolecular diagnostics have revolutionized the management of health care through enhanced detection of disease or infection and effective enrollment into treatment. In recognition of this, the World Health Organization approved the rollout of nucleic acid amplification technologies for identification ofMycobacterium tuberculosisusing platforms such as GeneXpert MTB/RIF, the GenoType MTBDRplusline probe assay, and, more recently, GeneXpert MTB/RIF Ultra. These assays can simultaneously detect tuberculosis infection and assess rifampin resistance. However, their widespread use in health systems requires verification and quality assurance programs. To enable development of these, we report the construction of genetically modified strains ofMycobacterium smegmatisthat mimic the profile ofMycobacterium tuberculosison both the GeneXpert MTB/RIF and the MTBDRplusline probe diagnostic tests. Using site-specific gene editing, we also created derivatives that faithfully mimic the diagnostic result of rifampin-resistantM. tuberculosis, with mutations at positions 513, 516, 526, 531, and 533 in the rifampin resistance-determining region of therpoBgene. Next, we extended this approach to other diseases and demonstrated that aStaphylococcus aureusgene sequence can be introduced intoM. smegmatisto generate a positive response for the SCCmecprobe in the GeneXpert SA Nasal Complete molecular diagnostic cartridge, designed for identification of methicillin-resistantS. aureus. These biomimetic strains are cost-effective, have low biohazard content, accurately mimic drug resistance, and can be produced with relative ease, thus illustrating their potential for widespread use as verification standards for diagnosis of a variety of diseases.

scholarly journals Improving the Sensitivity of the Xpert MTB/RIF Assay on Sputum Pellets by Decreasing the Amount of Added Sample Reagent: a Laboratory and Clinical Evaluation

2015 ◽  
Vol 53 (4) ◽  
pp. 1258-1263 ◽  
Author(s):  
Nila J. Dharan ◽  
Danielle Amisano ◽  
Gerald Mboowa ◽  
Willy Ssengooba ◽  
Robert Blakemore ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Smear Microscopy ◽  
Content Type ◽  
Clinical Specimens ◽  
Test Sensitivity ◽  
Smear Negative ◽  
Almost All ◽  
Culture Positive ◽  
Xpert Assay

The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection ofMycobacterium tuberculosisin sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions ofM. tuberculosisinto sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained fromM. tuberculosisculture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in whichM. tuberculosiswas detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%;P= 0.03) and for smear-negative specimens (67% versus 58%;P= 0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increasedM. tuberculosisdetection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.

scholarly journals Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay

2019 ◽  
Vol 58 (1) ◽  
Author(s):  
Yuan Cao ◽  
Heta Parmar ◽  
Ann Marie Simmons ◽  
Devika Kale ◽  
Kristy Tong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Melting Temperature ◽  
Gene Mutations ◽  
Test Sample ◽  
Rpob Gene ◽  
Automatic Identification ◽  
Content Type ◽  
Automated Algorithm ◽  
Wide Range ◽  
Rifampin Resistance

ABSTRACT Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific “Tm signature” reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

scholarly journals A 10-Year Comparative Analysis Shows that Increasing Prevalence of Rifampin-Resistant Mycobacterium tuberculosis in China Is Associated with the Transmission of Strains Harboring Compensatory Mutations

2018 ◽  
Vol 62 (4) ◽  
Author(s):  
Fengmin Huo ◽  
Jingjing Luo ◽  
Jin Shi ◽  
Zhaojing Zong ◽  
Wei Jing ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Comparative Analysis ◽  
Bacterial Population ◽  
Great Majority ◽  
Beijing Genotype ◽  
Isoniazid Resistance ◽  
Content Type ◽  
The Past ◽  
Rifampin Resistance ◽  
Compensatory Mutations

ABSTRACT In this work, we conducted bacterial population profile studies to assess trends of rifampin (RIF) resistance of Mycobacterium tuberculosis isolates collected across China from 2005 to 2015. Totals of 273 and 269 randomly selected M. tuberculosis isolates from 2005 and 2015, respectively, were analyzed. The rates of RIF resistance (36.4%), isoniazid resistance (39.0%), and levofloxacin resistance (25.7%) in 2015 were significantly higher than those in 2005 (28.2%, 30.0%, and 15.4%, respectively; P < 0.05). Genotypic data revealed 256 (95.2%) Beijing-type isolates in 2015, a rate significantly higher than that in 2005 (86.4%) ( P < 0.01). A higher proportion of mutations was identified within the rifampin resistance-determining region (RRDR) of rpoB in isolates from 2015 (99.0%) than in 2005 isolates (85.7%, P < 0.01). In addition, a significantly higher proportion of RIF-resistant isolates carrying compensatory mutations was observed in 2015 (31.6%) than in 2005 (7.8%). Notably, the great majority of these compensatory mutations (91.9%) were observed in isolates that harbored a mutation of codon 531 of the rpoB gene. In conclusion, our data demonstrate that resistance to RIF, isoniazid, and levofloxacin has become significantly more prevalent during the past decade. In addition, the prevalence of the Beijing genotype significantly increased from 2005 to 2015. Notably, a significantly increased frequency of strains with mutations in rpoC or rpoA is observed among those that have codon 531 mutations, which suggests that they may be compensatory and may play a role in facilitating transmission.

scholarly journals Comparison of Three Commercial Molecular Assays for Detection of Rifampin and Isoniazid Resistance among Mycobacterium tuberculosis Isolates in a High-HIV-Prevalence Setting: TABLE 1

2015 ◽  
Vol 53 (9) ◽  
pp. 3032-3034 ◽  
Author(s):  
K. Strydom ◽  
F. Ismail ◽  
M. M. Z. Matabane ◽  
O. Onwuegbuna ◽  
S. V. Omar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hiv Prevalence ◽  
Isoniazid Resistance ◽  
Molecular Assays ◽  
False Positivity ◽  
Version 2.0 ◽  
Rifampin Resistance ◽  
Xpert Assay

In a head-to-head comparison of the MTBDRplusversion 2.0 (Hain Lifescience), the Xpert MTB/RIF (Cepheid), and the Anyplex MTB/NTM (Seegene) assays, we demonstrated equal sensitivity (59/61; 96.7%) and specificity (53/54; 98.1%) for detecting rifampin resistance with further analysis of discordances. The Xpert assay does not detect isoniazid resistance while the Anyplex assay showed high false positivity.

scholarly journals Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China

2016 ◽  
Vol 60 (5) ◽  
pp. 2807-2812 ◽  
Author(s):  
Qin-jing Li ◽  
Wei-wei Jiao ◽  
Qing-qin Yin ◽  
Fang Xu ◽  
Jie-qiong Li ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Variable Number Tandem Repeat ◽  
Northern China ◽  
Multidrug Resistant ◽  
Variable Number ◽  
Beijing Genotype ◽  
Content Type ◽  
Resistant Tuberculosis ◽  
Rifampin Resistance

ABSTRACTMycobacterium tuberculosiscan acquire resistance to rifampin (RIF) through mutations in therpoBgene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in therpoAorrpoCgene. This study aimed to identifyrpoAandrpoCmutations in clinicalM. tuberculosisisolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found inrpoAorrpoCof the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in therpoA(n= 6) orrpoC(n= 83) gene. The proportion ofrpoCmutations was significantly higher in new cases (P= 0.023) and in strains with therpoBS531L mutation (P< 0.001). In addition, multidrug-resistant (MDR) strains withrpoCmutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat clustering (P= 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDRM. tuberculosisstrains.

Rifampin-resistance-associated mutations in the rifampin-resistance-determining region of the rpoB gene of Mycobacterium tuberculosis clinical isolates in Shanghai, PR China

2021 ◽  
Author(s):  
Yinjuan Guo ◽  
Xingwei Cao ◽  
Jinghui Yang ◽  
Xiaocui Wu ◽  
Yin Liu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Mutation Frequency ◽  
Rpob Gene ◽  
Clinical Isolates ◽  
Content Type ◽  
Link Type ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Rifampin Resistance

Introduction. Resistance to rifampin (RIF) in Mycobacterium tuberculosis infection is associated with mutations in the rpoB gene coding for the β-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive. Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance. Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against M. tuberculosis . Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for M. tuberculosis isolates were determined using commercial Sensititre M. tuberculosis MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis. Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01). Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by M. tuberculosis strains harbouring these mutations.

Sign in / Sign up

Export Citation Format

Share Document