Rifampin Resistance In Mycobacterium Tuberculosis Research Articles

Discordant results between Xpert MTB/RIF assay and Bactec MGIT 960 culture system regarding the detection of rifampin-resistant Mycobacterium tuberculosis isolates in Wenzhou, China.

This study aimed to assess the possible causes of discordant results between Xpert MTB/RIF (Xpert) and Bactec MGIT 960 Culture System (MGIT960) regarding rifampicin (RIF) susceptibility in Mycobacterium tuberculosis. Patients with previous RIF-resistant tuberculosis who were admitted to Wenzhou Central Hospital from January 2020 to December 2022 were enrolled. The isolates obtained from these patients were subjected to RIF susceptibility tests using Xpert and MGIT960, and the minimum inhibitory concentration (MIC) of RIF was determined by the MYCOTB MIC plate test. Additionally, molecular docking and molecular dynamics (MD) simulations were performed to evaluate the binding efficacy of rpoB and RIF based on rpoB mutations detected in the isolates with discordant RIF susceptibility results. A total of 28 isolates with discordant RIF susceptibility test results were detected, 15 of them were RIF susceptible with MICs ≤ 0.5 µg/mL. Twelve out of 15 isolates contained borderline RIF resistance-associated mutations [L430P (n = 6), H445N (n = 6)], 1 isolate had D435Y and Q429H double mutation, and the remaining 2 isolates had a silent (Q432Q) mutation. Compared with the affinity of RIF toward the wild type (WT) (-45.83 kcal/mol) by MD, its affinity toward L452P (-55.52 kcal/mol), D435Y (-47.39 kcal/mol), L430P (approximately -69.72 kcal/mol), H445N (-49.53 kcal/mol), and Q429H (-55.67 kcal/mol) increased. Borderline RIF resistance-associated mutations were the main cause for the discordant RIF susceptibility results between Xpert and MGIT960, and the mechanisms of the resistance need further investigated.IMPORTANCEThis study is aimed at assessing discordant results between Xpert MTB/RIF (Xpert) assay and Bactec MGIT 960 Culture System (MGIT960) regarding the detection of rifampicin (RIF)-resistant Mycobacterium tuberculosis isolates in Wenzhou, China. The discordant results of RIF between these two assays were mainly caused by borderline RIF resistance-associated mutations, subsequently by silent mutations of rpoB. Borderline RIF resistance- associated mutations detected in our study were demonstrated to not be affected by the affinity of rpoB and RIF by molecular dynamics, and the mechanism of resistance was needed to be clarified. For the discordant results of RIF by Xpert and MGIT960 that occurred, rpoB DNA sequencing was recommended to investigate its association with resistance to RIF.

Distribution of lineages and type II toxin-antitoxin systems among rifampin-resistant Mycobacterium Tuberculosis Isolates.

Type II toxin-antitoxin systems such as mazEF3, vapBC3, and relJK play a role in antibiotic resistance and tolerance. Among the different known TA systems, mazEF3, vapBC3, and relJK, which are type II systems, have specific roles in drug resistance. Therefore, the aim of this study was to investigate the mutations in these genes in sensitive and resistant isolates of Mycobacterium tuberculosis. Thirty-two rifampin-resistant and 121 rifampin-sensitive M. tuberculosis isolates were collected from various regions of Iran. Lineage typing was performed using the ASO-PCR method. Mutations in the rpoB gene were analyzed in all isolates by MAS-PCR. Furthermore, mutations in the mazEF3, relJK, and vapBC3 genes of the type II toxin system were assessed through PCR sequencing. These sequences were analyzed using COBALT and SnapGene 2017, and submitted to the GenBank database. Among the 153 M. tuberculosis samples, lineages 4, 3 and 2 were the most common. Lineage 2 had the highest rate of rifampin resistance. Mutations in rpoB531 were the most frequent in resistant isolates. Examination of the toxin-antitoxin system showed that rifampin-resistant isolates belonging to lineage 3 had mutations in either the toxin or antitoxin parts of all three TA systems. A mutation in nucleotide 195 (codon 65) of mazF3 leading to an amino acid change from threonine to isoleucine was detected in all rifampin-resistant isolates. M. tuberculosis isolates belonging to lineage 2 exhibited the highest rifampin resistance in our study. Identifying the mutation in mazF3 in all rifampin-resistant isolates can highlight the significance of this mutation in the development of drug resistance in M. tuberculosis. Expanding the sample size in future studies can help develop a new method for identifying resistant isolates.

Comparative evaluation of critical concentrationsfor detecting borderline rifampin resistance in Mycobacterium tuberculosis

Background: Rifampin plays an important role in tuberculosis treatment. In recent years, the introduction of molecular testing techniques has enabled the rapid detection of rifampin resistance, leading to discrepancies with conventional methods. The World Health Organization (WHO) has analyzed mutations in the rpoB gene that induce rifampin resistance and identified certain mutations causing borderline resistance, which are often undetected using conventional tests. Consequently, the WHO has lowered the rifampin resistance criterion concentration from 1.0 to 0.5 μg/mL in 7H10 and MGIT 960. The present study aimed to evaluate the impact of this change in critical concentration on the detection of borderline rifampin resistance in Mycobacterium tuberculosis. Methods: Tuberculosis strains submitted for antituberculosis drug susceptibility testing from May 2021–2022 were analyzed. Three institutions participated; the Seoul Clinical Laboratories used the agar proportion method, whereas the Samsung Medical Center and Seoul National University Bundang Hospital utilized the MGIT 960 system to test both the original and revised concentrations. Mutations were confirmed through rpoB gene sequencing for strains showing discrepancies. Results: A total of 1,596 valid susceptibility tests were conducted during the study period. Rifampin resistance was detected in 35 cases (2.19%) at 1.0 μg/mL and in 38 cases (2.38%) at 0.5 μg/mL, whereas isoniazid resistance was observed in 158 cases (9.90%). Among the three rifampin discrepancy strains, one harbored an H445L mutation, whereas the remaining two exhibited an L452P mutation. These mutations were classified as borderline resistant. Conclusion: Applying the new rifampin critical concentration resulted in a 0.19% increase in resistance rate and an 8.57% increase in detection cases. Additionally, despite testing with large number of rifampin-susceptible strains, no false resistance results were obtained. Therefore, the application of the new critical concentration is considered beneficial for the management of rifampin-resistant tuberculosis.

Molecular Approaches for Detection of Rifampin-Resistant Mycobacterium tuberculosis and Its Associated Risk Factors among General Population

GeneXpert MTB/RIF is a molecular technique for the diagnosis of tuberculosis. This method not only detects M. tuberculosis but also identifies the absence or presence of different drug resistant mutations in DNA fragments like rifampicin resistance. In this study, about 200 septum samples were collected from tuberculosis suspected people of Isfandyar Bukhari District Head Quarter Hospital Attock for screening and determination of Mycobacterium tuberculosis and multi-drug-resistant tuberculosis. The samples were screened for the presence of M. tuberculosis using Fluorescence Microscopy staining followed by GeneXpert MTB/RIF assay. The results were further confirmed by Line Probe Assay and Mycobacterium Growth Indicator Tube 960 techniques. In a total of 200 samples, 45(22.5%) were found positive, 155 (77.5%) were classified negative on culture while 38(19%) were positive and 162(81%) were negative on FM, 33(16.5%) were positive and 167(83.5%) were negative on ZN staining microscopy and 47(23.5%) were noted positive and 153 (76.5%) were negative on GeneXpert. The overall sensitivity of GeneXpert was 07% higher than that of smear microscopy and 4.2 % higher than culture.Different risk factors reported for TB are; malnutrition (23.40%), health care workers (14.89%), immunosuppression (12.76%), unimmunized with Bacille Calmette-Guérin (BCG) vaccine and contact with infected persons (10.63%), diabetes mellitus (8.51%), poverty and foreign-born children are (4.2%). It is concluded that the most common risk factor for TB was malnutrition (23.40%) and the foreign-born children (4.2%) were least affected. GeneXpert assay for identification of tuberculosis was found more sensitive than fluorescence microscopy and for isolation of MTB and rifampicin resistance, GeneXpert RIF assay can efficiently be usedwhich is simple, robust and efficient with minimum handling technique.

RpoB Mutations and Effects on Rifampin Resistance in Mycobacterium tuberculosis.

ObjectiveTo investigate the mutations within the whole rpoB gene of Mycobacterium tuberculosis and analyze their effects on rifampin (RIF) resistance based on crystal structure.MethodsWe sequenced the entire rpoB gene in 175 tuberculosis isolates and quantified their minimum inhibitory concentrations using microplate-based assays. Additionally, the structural interactions between wild-type/mutant RpoB and RIF were also analyzed.ResultsResults revealed that a total of 34 mutations distributed across 17 different sites within the whole rpoB gene were identified. Of the 34 mutations, 25 could alter the structural interaction between RpoB and RIF and contribute to RIF resistance. Statistical analysis showed that S450L, H445D, H445Y and H445R mutations were associated with high-level RIF resistance, while D435V was associated with moderate-level RIF resistance.ConclusionSome mutations within the rpoB gene could affect the interaction between RpoB and RIF and thus are associated with RIF resistance. These findings could be helpful to design new antibiotics and develop novel diagnostic tools for drug resistance in TB.

基于PARMS技术的结核分枝杆菌利福平耐药基因 rpoB 分子标记的开发

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

RpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China.

ObjectiveThe aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China.MethodsWe 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.ResultsA 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.ConclusionsThe 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.

Corrigendum to: Molecular Detection of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Isolates from Lorestan Province, Iran from 2014 to 2017

Corrigendum to: Molecular Detection of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Isolates from Lorestan Province, Iran from 2014 to 2017

Molecular Detection of Isoniazid and Rifampin Resistance in Mycobacterium tuberculosis Isolates from Lorestan Province, Iran from 2014 to 2017

Background: A rise in multidrug-resistant tuberculosis (MDR-TB), which is defined as the resistance to the two most effective first-line therapeutic drugs, Isoniazid (INH) and Rifampin (RIF), threatens global public health worldwide. Resistance of Mycobacterium tuberculosis to INH results from mutations in several genes most commonly in katG gene, and resistance to RIF is due to mutations in rpoB gene. Therefore, rapid diagnosis of MDR-TB is of high importance in controlling the disease progress and outcome. The accurate detection of the resistant TB strains can be accelerated by developing molecular tests. Objectives: The aim of the present local study was to isolate MDR-TB from the patients who were the residents in the west of Iran and examination the frequency of MDR-TB between patients of Lorestan province for the first time and assess the mutations in the regions related to RIF/INH resistance via PCR and sequencing methods. Methods: In this study, 106 isolates of M. tuberculosis were selected in health centers of Lorestan, Iran from 2014 to 2017. After culturing M. tuberculosis isolates on Lowenstein-Jensen medium, classical susceptibility testing proportional method against INH and RIF was performed. After DNA extraction, PCR and sequencing were used to detect mutations related to RIF and INH resistance. Results: The results demonstrated 3.8%, 0.9%, and 0.9% frequency for INH + RIF, INH and RIF resistance, respectively. Importantly, 4 out of 6 isolates harbored mutations in codon290 of katG gene. Also, these isolates contained mutations in codon339 of rpoB gene. No mutation was observed in inhA gene of M. tuberculosis isolates. Conclusions: The results suggest that molecular techniques can be used as a rapid method for the identification of drug resistance in clinical isolates of M. tuberculosis. DNA sequencing has a high sensitivity for the detection of resistance mutations to RIF and INH in MDR-TB cases. Also, the results showed that the most frequent resistance associated-mutations occurred in codon290 of katG and codon 339 rpoB gene segments.

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.

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.

A Diagnostic Algorithm To Investigate Pyrazinamide and Ethambutol Resistance in Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Low-Incidence Setting.

Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug-resistant Mycobacterium tuberculosis complex isolates. Sequencing results were validated using whole-genome sequencing (WGS) of the isolates. Resistance-conferring mutations obtained by pncA sequencing correlated well with phenotypic DST results for pyrazinamide. Phenotypic resistance to ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance-conferring mutations were found in the embB gene at codons 354, 406, and 497. In several isolates that tested ethambutol susceptibility by phenotypic DST, well-known resistance-conferring embB mutations were determined. Thus, targeted Sanger sequencing beyond the embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility testing, as a basis for the rational design of multidrug-resistant tuberculosis regimens with or without ethambutol.

Direct Detection of Rifampin-Resistant Mycobacterium tuberculosis in Respiratory Specimens Using Quantamatrix Multiplexed Assay Platform (QMAP) System: A Multicenter Study in Korea.

Rapid and accurate detection of rifampin-resistant Mycobacterium tuberculosis (MTB) is of primary importance for infection control and selection of anti-tuberculosis drugs. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the direct detection of rifampin-resistant MTB in respiratory specimens. A total of 400 respiratory specimens collected from patients with clinically suspected tuberculosis or non-tuberculous mycobacteria (NTM) infections were tested with the culture-based conventional Mycobacterium species identification and QMAP system. Among 400 specimens, 154 samples were evaluated using phenotypic anti-tuberculosis drug susceptibility test (DST) and the QMAP system for the detection of rifampin resistance. Detection agreement rate between the culture-based conventional identification and QMAP system for MTB and NTM according to acid-fast bacillus smear positivity was as follows: 97.0% (131/135) and 93.6% (88/94) in 229 smear-positive samples and 69.4% (25/36) and 73.0% (65/89) in 171 smear-negative samples. Based on culture as the gold standard, the overall sensitivity and specificity of the QMAP system for Mycobacterium identification were 87.3 and 97.8%, respectively. The categorical agreement rate between phenotypic DST and QMAP system for rifampin was as follows: complete agreement, 92.9% (143/154); very major error, 0%; and major error, 0.6% (1/154). The overall sensitivity of the QMAP system for the detection of rifampin resistance was 97.1% (34/35). The QMAP system is a useful screening method for the early diagnosis of tuberculosis and selection of anti-tuberculosis drug, as it may detect rifampin-resistant MTB directly from respiratory specimens.

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.

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.

Direct detection of Mycobacterium tuberculosis rifampin resistance in bio-safe stained sputum smears.

Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing.

Clinical Impact of Rpob Mutations Conferring Discordant Phenotypic-Genotypic Rifampin Resistance in Mycobacterium Tuberculosis

isolates tested RIF susceptible phenotypically with a missense mutation in rpoB, were included in this study. • Pyrosequencing was used to determine rpoB mutations. • Demographic data abstracted from California TB registry. • Clinical information including medical history, TB treatment, and outcomes were abstracted from medical records. • Treatment outcomes defined by World Health Organization were used. • Rifampin (RIF) is the cornerstone of TB treatment; when RIF cannot be used, treatment duration is prolonged. • Accurate RIF testing is essential. • Phenotypic drug susceptibility testing (DST) is current gold standard, however, molecular assays provide rapid DSTs results leading to a decreased time to treatment imitation. • Limited data suggests that isolates with rpoB mutations that test phenotypically susceptible to RIF may have clinical significance. • However, data were from international settings where management of TB differs from the U.S. Clinical impact of rpoB mutations conferring discordant phenotypic-genotypic rifampin resistance in Mycobacterium tuberculosis N. Shah1,2, SYG. Lin2, P.M. Barry2, G. Schecter2, E. Desmond2 1Centers for Disease Control and Prevention, Atlanta, GA; 2California Department of Public Health, Richmond, CA Background

Predicting differential rifamycin resistance in clinical Mycobacterium tuberculosis isolates by specific rpoB mutations.

Rifampin (RMP) resistant Mycobacterium tuberculosis is usually assumed to be resistant to all rifamycins. Increasing evidence indicates, however, that some rpoB mutations, detectable by rapid molecular diagnostics, confer resistance to RMP but not to rifabutin (RBT), suggesting that RBT may be effective for the treatment of M. tuberculosis with these mutations. To determine if specific rpoB mutations reliably predict differential phenotypic resistance to RMP and RBT. We selected 60 clinical M. tuberculosis isolates from a repository of multinational multidrug-resistant tuberculosis isolates and stratified them into two groups: 1) those with rpoB mutations suspected to confer differential resistance to RMP and RBT, and 2) those expected to be cross-resistant to RMP and RBT. These assumptions were tested by comparing the phenotypic susceptibilities of RMP/RBT with those predicted by mutations in the rpoB gene. Of 20 suspected RMP-resistant/RBT-susceptible isolates, 15 were RMP-resistant but RBT-susceptible, 3 were RMP- and RBT-susceptible, and 2 were cross-resistant to both RMP and RBT. In comparison, 40 of 40 suspected cross-resistant isolates were both RMP- and RBT-resistant. Our data support the association between specific rpoB mutations and differential resistance of M. tuberculosis to RMP and RBT. Clinical studies are required to investigate the efficacy of RBT in the treatment of M. tuberculosis harboring these mutations.

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

Among 452 samples that were positive by the Xpert MTB/RIF (Xpert) assay and MGIT 960 system (MGIT), 440 and 10 Mycobacterium tuberculosis samples 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. rpoB sequencing 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 the rpoB gene.

Evaluation of the RNA simultaneous amplification and testing for detection of rifampin resistance in Mycobacterium tuberculosis

To establish and evaluate a method for detection of rifampin resistance in Mycobacterium tuberculosis (M. tuberculosis) by the RNA simultaneous amplification and testing (SAT). RNA probe and primer of reverse transcription with T7 promoter targets pre-16S rRNA were designed, and the isothermal RNA amplification at 42 °C was performed for real-time detection of the levels of pre-16S rRNA in drug exposed MTB. Twenty clinical isolates were detected by the SAT after treatment with rifampicin 1, 2 and 3 d in order to determine the best drug effect time. Fifty clinical isolates with known drug susceptibility results were used to determine the best cutoff value of rifampicin drug susceptibility by SAT. In total, 128 clinical isolates were detected by SAT to evaluate the accuracy compared with the Bactec MGIT 960. The best drug effect time and cutoff value for the detection of rifampin resistance by SAT were 2 d and 2.95. With the result of Bactec MGIT 960 as the reference, the sensitivity and the specificity of the assay was 100% (51/51) and 97.4% (75/77), respectively. The isothermal RNA amplification assay is a highly sensitive and specific tool for the detection of rifampicin resistance in M. tuberculosis, and therefore, it may be a new method to detect rifampicin resistance in M. tuberculosis.

Development of a single multiplex amplification refractory mutation system PCR for the detection of rifampin-resistant Mycobacterium tuberculosis

A rapid and simple method for the detection of drug-resistant Mycobacterium tuberculosis is critical for the efficient treatment and control of this pathogen in developing country. Here we developed a single multiplex amplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer and temperature switch PCR (TSP) strategy were included. Using this method, we detected rifampin resistance-associated mutations at codons 511, 516, 526 and 531 in the rifampin resistance-determining region of rpoB gene. The performance of M-ARMS-PCR assay was evaluated with 135 cultured isolates of M. tuberculosis. The sensitivity and specificity were 94.2% and 100%, respectively, compared with direct DNA sequencing, and 86.67% and 89.71%, respectively, compared with culture-based phenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCR method is useful and efficient with an intended application in provincial Centers for Disease Control and Prevention for rapid detection of rifampin resistance-associated mutations.

X-ray Crystal Structures of the Escherichia coli RNA Polymerase in Complex with Benzoxazinorifamycins

Rifampin, a semisynthetic rifamycin, is the cornerstone of current tuberculosis treatment. Among many semisynthetic rifamycins, benzoxazinorifamycins have great potential for TB treatment due to their superior affinity for wild-type and rifampin-resistant Mycobacterium tuberculosis RNA polymerases and their reduced hepatic Cyp450 induction activity. In this study, we have determined the crystal structures of the Escherichia coli RNA polymerase complexes with two benzoxazinorifamycins. The ansa-naphthalene moieties of the benzoxazinorifamycins bind in a deep pocket of the β subunit, blocking the path of the RNA transcript. The C3'-tail of benzoxazinorifamycin fits a cavity between the β subunit and σ factor. We propose that in addition to blocking RNA exit, the benzoxazinorifamycin C3'-tail changes the σ region 3.2 loop position, which influences the template DNA at the active site, thereby reducing the efficiency of transcription initiation. This study supports expansion of structure-activity relationships of benzoxazinorifamycins inhibition of RNA polymerase toward uncovering superior analogues with development potential.