Characterization of rpoB Mutations in Rifampin-Resistant Clinical Mycobacterium tuberculosis Isolates from Greece

There is a geographic distribution of Mycobacterium tuberculosis strains with various rpoB gene mutations that account for rifampin resistance. We studied 17 rifampin-resistant clinical isolates from patients in Greece to identify rpoB mutations. The aim of our study was the evaluation of a commercially available line probe assay kit (INNO-LiPA Rif. TB) to detect rpoB mutations and rifampin resistance. The results obtained with the commercially available assay were compared to those obtained by automated DNA sequence analysis of amplified PCR products. Randomly amplified polymorphic DNA (RAPD) analyses of the isolates were also performed. The overall concordance of the line probe assay with phenotypic rifampin susceptibility test was 94%. Three distinct rpoB mutations in codons Ser₅₃₁, His₅₂₆, and Asp₅₁₆ were correctly identified with the kit, but mutations in external regions and insertions were detected only by automated DNA sequence analysis. The changes in codons Ser₅₃₁ and His₅₂₆ accounted for the majority of rifampin resistance, as previously described for isolates from other geographic areas. The results obtained by RAPD analyses of the isolates suggested that clonally related M. tuberculosis strains can have subclones bearing distinct mutant rpoB alleles. We conclude that this line probe assay kit, which is fast and with which tests are easy to perform, can be used for the rapid detection of rifampin resistance in M. tuberculosis before the availability of results by conventional methods and for epidemiological studies but that negative results obtained by this method do not rule out rifampin resistance.     

Mycobacterium Species Identification and Rifampin Resistance Testing with High-Density DNA Probe Arrays

Species identification within the genus Mycobacterium and subsequent antibiotic susceptibility testing still rely on time-consuming, culture-based methods. Despite the recent development of DNA probes, which greatly reduce assay time, there is a need for a single platform assay capable of answering the multitude of diagnostic questions associated with this genus. We describe the use of a DNA probe array based on two sequence databases: one for the species identification of mycobacteria (82 unique 16S rRNA sequences corresponding to 54 phenotypical species) and the other for detecting Mycobacterium tuberculosis rifampin resistance (rpoB alleles). Species identification or rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Seventy mycobacterial isolates from 27 different species and 15 rifampin-resistant M. tuberculosis strains were tested. A total of 26 of 27 species were correctly identified as well as all of the rpoB mutants. This parallel testing format opens new perspectives in terms of patient management for bacterial diseases by allowing a number of genetic tests to be simultaneously run.     

Rapid detection of rpoB mutations in rifampin resistant M. tuberculosis from sputum samples by denaturing gradient gel electrophoresis

Objective: To establish a rapid detection method for identifying rpoB mutations associated with rifampin (RIF) resistance in sputum specimens.Methods: We detected rpoB mutations directly in 90 sputum specimens collected from suspected tuberculosis patients using PCR-based denaturing gradient gel electrophoresis (DGGE) and compared these results with those obtained by rpoB sequencing and conventional drug susceptibility testing.Results: The positive detection rate of Mycobacterium tuberculosis (M. tuberculosis) was 52.2% by Acid-Fast Bacilli staining and 72.2% by conventional mycobacterial culture. In contrast, the positive rate was significantly higher (93.3%) by PCR-based detection of the rpoB gene in the same specimens. Furthermore, 75% of the tested specimens presented abnormal patterns compared with the wild-type pattern (standard H37Rv strain) analysed by DGGE. A total of 12 different patterns, representing 12 different rpoB mutations, were observed in the 63 abnormal patterns. The match rate of rpoB mutations detected by DGGE reached 96.9% when compared to DNA sequencing.Conclusion: Our findings indicate that PCR-based DGGE is a rapid and reliable bio-technique for direct detection of rpoB mutations associated with RIF resistance in the sputum of suspected tuberculosis patients.     

Detection of rpoB mutations in Mycobacterium tuberculosis by biprobe analysis

A biprobe assay utilizing LightCycler technology was developed to detect rifampin resistance-associated gene mutations in the Mycobacterium tuberculosis rpoB gene. Three biprobes detected all mutations present in the 46 rifampin-resistant isolates. Wild-type sequences were correctly identified in each case. The method was reproducible, accurate, and easy to use.     

Detection of Mycobacterium tuberculosis resistance mutations to rifampin and isoniazid by real-time PCR

Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR)-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible) using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100%) and 95% (95% CI: 89% to 100%), respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100%) and 74% (95% CI: 61% to 87%), respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.     

Detection of rpoB,katG and inhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqing as determined by microarray

The emergence of multidrug-resistance Mycobacterium tuberculosis is an increasing threat to tuberculosis control programmes. Susceptibility testing of Mycobacterium tuberculosis complex isolates by traditional methods requires a minimum of 14 days. This can be reduced significantly if molecular analysis is used. DNA sequencing is a good method for detecting mutation, but cannot be used routinely because of its relatively high cost. A sensitive and specific microarray has been designed to detect mutations in the rifampin resistance determining region of rpoB and loci in katG and inhA associated with isoniazid (INH) resistance. A panel of Mycobacterium tuberculosis isolates containing 13 different rpoB genotypes, two mutation genotypes within codon 315 of katG and one mutation genotypes at inhA was used to validate the microarray. The results obtained indicate that 100% of rifampicin-resistant M. tuberculosis strains isolated in Chongqing had rpoB mutations, with 531-Ser and 526-His being the most common positions substituted. Of the total 50 INH resistant isolates, 82% had a katG315 mutation and 18% had an inhA mutation. All the mutations detected by the microarray method were also confirmed by conventional DNA sequencing. It is demonstrated that the microarray is an efficient, specialized technique and can be used as a rapid method for detecting rifampin and isoniazid resistance.     

Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin,Isoniazid, and Fluoroquinolones

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.Rifampin (rifampicin), isoniazid, and the fluoroquinolones are the most important initial drug markers for extensively drug-resistant Mycobacterium tuberculosis strains, defined as multidrug-resistant (MDR) isolates (resistant to both isoniazid and rifampin) with additional resistance to a fluoroquinolone and to one of the injectable drugs (2). The fluoroquinolones have become an essential part of treatment regimens for MDR tuberculosis (7, 25). Due to their potency and safety, the new-generation fluoroquinolones are now even being evaluated as first-line medications for tuberculosis (3, 13, 20). Wang et al. further suggested that routine fluoroquinolone resistance testing may have a clinical impact by showing a significant correlation between development of fluoroquinolone and first-line M. tuberculosis drug resistance in an area in which resistant strains are highly endemic (28).The spontaneous acquisition of DNA sequence mutations is the primary genetic basis for the development of M. tuberculosis drug resistance (14). Since sequences are highly conserved, certain mutations correlate well with phenotypic resistance, and a limited number of mutations account for the majority of phenotypic resistance to the important antituberculosis medications, various methods of genotypic testing have successfully been used for the rapid detection of M. tuberculosis resistance (16, 22). The sites that most frequently contain mutations associated with phenotypic resistance, called resistance-determining regions (RDR), differ depending on the drug tested. Among rifampin-resistant isolates worldwide, 95 to 97% harbor mutations in the rifampin RDR, an 81-bp target encompassing codons 507 to 533 of the 3,519-bp rpoB gene (17). Isoniazid resistance has a more complex mechanism, involving several gene targets, the most important of which is codon 315 of the 2,223-bp katG gene, in which mutations are found in up to 50% of resistant isolates (18). Likewise, M. tuberculosis has a quinolone RDR which spans codons 88 to 94 of the 2,517-bp gyrA gene. Mutations in this region, particularly in codon 88, 90, 91, or 94, correlate with high-level resistance and are seen in 42 to 85% of resistant clinical isolates (6).Pyrosequencing, a method of DNA sequencing by synthesis, has been applied to the rapid detection of M. tuberculosis resistance to rifampin, isoniazid, and ethambutol (9, 29). Its main advantage is a much shorter turnaround time than that of conventional drug susceptibility testing, the latter taking 2 to 4 weeks from the time an isolate is obtained in pure culture.After isoniazid and rifampin, the next pivotal drug class in M. tuberculosis treatment is the fluoroquinolone class, as previously discussed (3, 7, 13, 20, 25, 28). Given that most resistance to the latter is determined by mutations that are generally limited to the quinolone RDR of the gyrA gene, it should be feasible and clinically more relevant to develop an assay for rapid resistance testing which includes fluoroquinolone resistance in addition to rifampin and isoniazid resistance.We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones, which we validated against the conventional submerged-disk proportion method. We also improved on the previously reported pyrosequencing assay by reducing the number of primers required to sequence for rifampin resistance (9).     

Rapid and accurate detection of rifampin and isoniazid-resistant Mycobacterium tuberculosis using an oligonucleotide array

To rapidly detect rifampin, isoniazid and multidrug resistance in Mycobacterium tuberculosis isolates, a new system (BluePoint MtbDR, Bio Concept Inc., Taichung, Taiwan) including an oligonucleotide array and an automatic reader was evaluated. The array simultaneously identifies M. tuberculosis and predominant mutations in the rpoB, katG and inhA upstream regulatory region (inhA-r) genes. The system was assessed with 324 clinical M. tuberculosis isolates, including 210 multidrug-resistant, 41 rifampin mono-resistant, 34 isoniazid mono-resistant and 39 fully susceptible isolates. The results were compared with those obtained using the GenoType MTBDRplus test, drug-resistant gene sequencing and conventional drug susceptibility testing. The detection limit of the array was 25 pg DNA. The array and the GenoType MTBDRplus test detected 179 (85.2%) and 182 (86.7%) multidrug-resistant M. tuberculosis strains, respectively. The sensitivities of the array for detecting rifampin and isoniazid resistance were 98.4% and 87.7%, respectively, whereas the sensitivities of the GenoType MTBDRplus test for detecting rifampin and isoniazid resistance were 98.8% and 88.9%, respectively. No significant difference was found between the tests with respect to their sensitivities to detect multidrug resistance (p 0.66), rifampin resistance (p 0.69) or isoniazid resistance (p 0.68). The discrepancies were mainly attributed to rare mutations in inhA-r, which were not included in the array. The array can directly reveal transmission-associated mutations, which are useful for epidemiological investigations. The turnaround time of the array test was 6–7 h. This study confirms the feasibility of using this system for rapid and accurate diagnosis of isoniazid and rifampin resistance in M. tuberculosis.     

Bacillus Calmette–Guérin strains with defined resistance mutations: a new tool for tuberculosis laboratory quality control

ObjectiveLaboratory quality control (QC) is essential to assess the reliability of tuberculosis diagnostic testing. To provide safe QC reagents for the detection of drug-resistant Mycobacterium tuberculosis, we generated antibiotic-resistant mycobacterial strains of attenuated virulence (M. bovis bacillus Calmette–Guérin (BCG)).MethodsSeven mono-resistant BCG strains were developed by introducing resistance-conferring mutations into wild-type BCG strains. Mutations were confirmed by dideoxynucleotide sequencing. Phenotypic resistance was quantified by microbroth dilution to determine the MIC₉₀. The capacity of two commercial tests (GeneXpert TB/RIF and Genotype MTBDRplus) to detect resistance-conferring mutations was evaluated independently.ResultsOur panel included BCG strains with mutations in rpoB (S450L, I491F), katG (deletion at AA428), gyrA (D94G), rpsL (K43R) and Rv0678c (S63R). These mutations translated respectively into phenotypic resistance to rifampin (MIC ≥8 mg/L), isoniazid (MIC ≥8 mg/L), moxifloxacin (MIC 4 mg/L) and streptomycin (MIC ≥8 mg/L); the Rv0678c mutant showed decreased susceptibility to both clofazimine (MIC 4 mg/L) and bedaqualine (MIC 1 mg/L). GeneXpert (Cepheid) and Genotype MTBDRplus (Hain Lifesciences) both called the rpoB S450L strain rifampin-resistant and the I491F mutant rifampin-susceptible, as expected based on single nucleotide polymorphism positions. Likewise, MTBDRplus called the novel katG deletion mutant isoniazid susceptible despite phenotypic resistance.ConclusionBCG strains engineered to be mono-resistant to anti-tuberculosis drugs can be used as safe QC reagents for tuberculosis diagnostics and drug susceptibility testing.     

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.     

Profiling of rpoB Mutations and MICs for Rifampin and Rifabutin in Mycobacterium tuberculosis

Resistance to rifampin (RIF) and rifabutin (RFB) in Mycobacterium tuberculosis is associated with mutations within an 81-bp region of the rpoB gene (RIF resistance-determining region [RRDR]). Previous studies have shown that certain mutations in this region are more likely to confer high levels of RIF resistance, while others may be found in phenotypically susceptible isolates. In this study, we sought to determine the relationship between the MICs of RIF and RFB and rpoB RRDR mutations in 32 multidrug-resistant (MDR), 4 RIF-monoresistant, and 5 susceptible M. tuberculosis clinical isolates. The MICs were determined using the MGIT 960 system. Mutations in the rpoB RRDR were determined by Sanger sequencing. RpoB proteins with mutations S531L (a change of S to L at position 531), S531W, H526Y, and H526D and the double mutation D516A-R529Q were associated with high MICs for RIF and RFB. Five isolates carrying the mutations L511P, H526L, H526N, and D516G-S522L were found to be susceptible to RIF. Several mutations were associated with resistance to RIF and susceptibility to RFB (F514FF, D516V, and S522L). Whole-genome sequencing of two MDR isolates without rpoB RRDR mutations revealed a mutation outside the RRDR (V146F; RIF MIC of 50 μg/ml). The implications of the polymorphisms identified in the second of these isolates in RIF resistance need to be further explored. Our study further establishes a correlation between the mutations and the MICs of RIF and, also, RFB in M. tuberculosis. Several rpoB mutations were identified in RIF- and RFB-susceptible isolates. The clinical significance of these findings requires further exploration. Until then, a combination of phenotypic and molecular testing is advisable for drug susceptibility testing.     

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis

Background: Early detection of multidrug-resistant tuberculosis (MDR-TB) is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. Materials and Methods: High-resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R), 21 isoniazid resistant (INH-R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates) and katG315 (85.7% of INH-R isolates), respectively. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.     

Point-of-Care System for Detection of Mycobacterium tuberculosis and Rifampin Resistance in Sputum Samples

Early detection of Mycobacterium tuberculosis complex (MTBC) and markers conveying drug resistance can have a beneficial impact on preventive public health actions. We describe here a new molecular point-of-care (POC) system, the Genedrive, which is based on simple sample preparation combined with PCR to detect MTBC and simultaneously detect mutation markers in the rpoB gene directly from raw sputum sample. Hybridization probes were used to detect the presence of the key mutations in codons 516, 526, and 531 of the rpoB gene. The sensitivities for MTBC and rpoB detection from sputum samples were assessed using model samples spiked with known numbers of bacteria prepared from liquid cultures of M. tuberculosis. The overall sensitivities were 90.8% (95% confidence interval [CI], 81, 96.5) for MTBC detection and 72.3% (95% CI, 59.8, 82.7) for rpoB detection. For samples containing ≥1,000 CFU/ml, the sensitivities were 100% for MTBC and 85.7% for rpoB detection, while for samples containing ≤100 CFU/ml, the sensitivities were 86.4% and 65.9% for MTBC and rpoB detection, respectively. The specificity was shown to be 100% (95% CI, 83.2, 100) for MTBC and rpoB. The clinical sputum samples were processed using the same protocol and showed good concordance with the data generated from the model. Tuberculosis-infected subjects with smear samples assessed as scanty or negative were detectable by the Genedrive system. In these paucibacillary patients, the performance of the Genedrive system was comparable to that of the GeneXpert assay. The characteristics of the Genedrive platform make it particularly useful for detecting MTBC and rifampin resistance in low-resource settings and for reducing the burden of tuberculosis disease.     

Development of multiplex assay for rapid characterization of Mycobacterium tuberculosis

We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB, katG, inhA, embB), members of the M. tuberculosis complex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG, gyrA), and Haarlem and Beijing strains (ogt, mutT2, mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.     

Rifampin Drug Resistance Tests for Tuberculosis: Challenging the Gold Standard

The rapid diagnosis of rifampin resistance is hampered by a reported insufficient specificity of molecular techniques for detection of rpoB mutations. Our objective for this study was to document the prevalence and prognostic value of rpoB mutations with unclear phenotypic resistance. The study design entailed sequencing directly from sputum of first failure or relapse patients without phenotypic selection and comparison of the standard retreatment regimen outcome, according to the mutation present. We found that among all rpoB mutations, the best-documented “disputed” rifampin resistance mutations (511Pro, 516Tyr, 526Asn, 526Leu, 533Pro, and 572Phe) made up 13.1% and 10.6% of all mutations in strains from Bangladesh and Kinshasa, respectively. Except for the 511Pro and 526Asn mutations, most of these strains with disputed mutations tested rifampin resistant in routine Löwenstein-Jensen medium proportion method drug susceptibility testing (DST; 78.7%), but significantly less than those with common, undisputed mutations (96.3%). With 63% of patients experiencing failure or relapse in both groups, there was no difference in outcome of first-line retreatment between patients carrying a strain with disputed versus common mutations. We conclude that rifampin resistance that is difficult to detect by the gold standard, phenotypic DST, is clinically and epidemiologically highly relevant. Sensitivity rather than specificity is imperfect with any rifampin DST method. Even at a low prevalence of rifampin resistance, a rifampin-resistant result issued by a competent laboratory may not warrant confirmation, although the absence of a necessity for confirmation needs to be confirmed for molecular results among new cases. However, a result of rifampin susceptibility should be questioned when suspicion is very high, and further DST using a different system (i.e., genotypic after phenotypic testing) would be fully justified.     

Evaluation of the Analytical Performance of the Xpert MTB/RIF Assay

We performed the first studies of analytic sensitivity, analytic specificity, and dynamic range for the new Xpert MTB/RIF assay, a nucleic acid amplification-based diagnostic system that detects Mycobacterium tuberculosis and rifampin (RIF) resistance in under 2 h. The sensitivity of the assay was tested with 79 phylogenetically and geographically diverse M. tuberculosis isolates, including 42 drug-susceptible isolates and 37 RIF-resistant isolates containing 13 different rpoB mutations or mutation combinations. The specificity of the assay was tested with 89 nontuberculosis bacteria, fungi, and viruses. The Xpert MTB/RIF assay correctly identified all 79 M. tuberculosis isolates and correctly excluded all 89 nontuberculosis isolates. RIF resistance was correctly identified in all 37 resistant isolates and in none of the 42 susceptible isolates. Dynamic range was assessed by adding 10² to 10⁷ CFU of M. tuberculosis into M. tuberculosis-negative sputum samples. The assay showed a log-linear relationship between cycle threshold and input CFU over the entire concentration range. Resistance detection in the presence of different mixtures of RIF-resistant and RIF-susceptible DNA was assessed. Resistance detection was dependent on the particular mutation and required between 65% and 100% mutant DNA to be present in the sample for 95% certainty of resistance detection. Finally, we studied whether assay specificity could be affected by cross-contaminating amplicons generated by the GenoType MTBDRplus assay. M. tuberculosis was not detected until at least 10⁸ copies of an MTBDRplus amplicon were spiked into 1 ml of sputum, suggesting that false-positive results would be unlikely to occur.Conventional diagnostic methods for Mycobacterium tuberculosis are slow and/or lack sensitivity. A number of new diagnostic approaches have brought incremental improvements to detection and drug susceptibility testing; however, the technical complexity of these assays and their dependence on dedicated laboratory infrastructure have limited their adoption, especially in low-resource, high-burden settings (1, 11, 12, 21). The recently introduced Xpert MTB/RIF (manufactured and marketed by Cepheid, Sunnyvale, CA) assay simultaneously detects the presence of M. tuberculosis and its susceptibility to the important first-line drug rifampin (RIF) (7). A sample processing system and an automated heminested real-time PCR assay are integrated into a single disposable cartridge. The assay can be performed directly from a clinical sputum sample or from a decontaminated sputum pellet and can generally be completed in less than 2 h (7).The Xpert MTB/RIF assay detects M. tuberculosis and RIF resistance by PCR amplification of the rifampin resistance-determining region (RRDR) of the M. tuberculosis rpoB gene and subsequent probing of this region for mutations that are associated with RIF resistance. Approximately 95% of RIF-resistant tuberculosis cases contain mutations in this 81-bp region (16). Our previous work has established that the Xpert MTB/RIF assay has a limit of detection (LOD), defined as the minimum number of bacilli that can be detected with 95% confidence) of 131 CFU per ml of clinical sputum (7). The assay was also able to identify RIF resistance in samples containing 23 common clinically occurring rpoB mutations. None of the 20 nontuberculosis mycobacteria (NTM) species tested, including the NTM species commonly described as causing human disease were falsely identified as M. tuberculosis (7), suggesting high specificity. Several small studies using clinical samples demonstrated 98% to 100% sensitivity overall, 72% sensitivity in smear-negative patients, and a specificity of 100% (7).In the present study, we expand upon our previous work and report on several critical analytical assay performance characteristics, including dynamic range, sensitivity, specificity, RIF resistance detection in heterogeneous samples, and resiliency against cross-contamination by other nucleic acid amplification techniques (NAATs).     

Rifampin Resistance Missed in Automated Liquid Culture System for Mycobacterium tuberculosis Isolates with Specific rpoB Mutations

WHO-endorsed phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are assumed to be the gold standard for identifying rifampin (RMP) resistance. However, previous results indicated that low-level, yet probably clinically relevant, RMP resistance linked to specific rpoB mutations is easily missed by some growth-based methods. We aimed to compare the level of resistance detected on Löwenstein-Jensen (LJ) medium with resistance detected by the Bactec MGIT 960 automated DST (MGIT-DST) system for various rpoB mutants. Full agreement between LJ and MGIT-DST was observed for mutations located at codons 513 (Lys or Pro) and 531 (Leu, Trp), which were always resistant by both methods. For mutations 511Pro, 516Tyr, 533Pro, 572Phe, and several 526 mutations, LJ and MGIT results were highly discordant, with MGIT-DST failing to give a result or declaring the strains susceptible. Our data show that phenotypic RMP resistance testing of M. tuberculosis is not a binary phenomenon for some rpoB mutations and that the widely used automated MGIT 960 system is prone to miss some RMP resistance-conferring mutations, while careful DST on LJ missed hardly any. Given the association of these mutations with poor clinical outcome, our findings suggest that the gold standard for rifampin resistance should be reconsidered, in order to address the present confusion caused by discrepancies between phenotypic and genotypic results. The impacts of these mutations will depend on the frequency of their occurrence, which may vary from one setting to another.     

Rapid, high-throughput detection of rifampin resistance and heteroresistance in Mycobacterium tuberculosis by use of sloppy molecular beacon melting temperature coding

Rifampin resistance in Mycobacterium tuberculosis is largely determined by mutations in an 80-bp rifampin resistance determining region (RRDR) of the rpoB gene. We developed a rapid single-well PCR assay to identify RRDR mutations. The assay uses sloppy molecular beacons to probe an asymmetric PCR of the M. tuberculosis RRDR by melting temperature (T(m)) analysis. A three-point T(m) code is generated which distinguishes wild-type from mutant RRDR DNA sequences in approximately 2 h. The assay was validated on synthetic oligonucleotide targets containing the 44 most common RRDR mutations. It was then tested on a panel of DNA extracted from 589 geographically diverse clinical M. tuberculosis cultures, including isolates with wild-type RRDR sequences and 25 different RRDR mutations. The assay detected 236/236 RRDR mutant sequences as mutant (sensitivity, 100%; 95% confidence interval [CI], 98 to 100%) and 353/353 RRDR wild-type sequences as wild type (specificity, 100%; 95% CI, 98.7 to 100%). The assay identified 222/225 rifampin-resistant isolates as rifampin resistant (sensitivity, 98.7%; 95% CI, 95.8 to 99.6%) and 335/336 rifampin-susceptible isolates as rifampin susceptible (specificity, 99.7%; 95% CI, 95.8 to 99.6%). All mutations were either individually identified or clustered into small mutation groups using the triple T(m) code. The assay accurately identified mixed (heteroresistant) samples and was shown analytically to detect RRDR mutations when present in at least 40% of the total M. tuberculosis DNA. This was at least as accurate as Sanger DNA sequencing. The assay was easy to use and well suited for high-throughput applications. This new sloppy molecular beacon assay should greatly simplify rifampin resistance testing in clinical laboratories.     

Evaluation of the AID TB Resistance Line Probe Assay for Rapid Detection of Genetic Alterations Associated with Drug Resistance in Mycobacterium tuberculosis Strains

The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected resistance mutations in clinical M. tuberculosis isolates from areas with low and high levels of endemicity (Switzerland, n = 104; South Africa, n = 52) and in selected Mycobacterium bovis BCG 1721 mutant strains (n = 5) with 100% accuracy. Subsequently, the line probe assay was shown to be capable of rapid genetic assessment of drug resistance in MGIT broth cultures, the results of which were in 100% agreement with those of DNA sequencing and phenotypic drug susceptibility testing. Finally, the line probe assay was assessed for direct screening of smear-positive clinical specimens. Screening of 98 clinical specimens demonstrated that the test gave interpretable results for >95% of them. Antibiotic resistance mutations detected in the clinical samples were confirmed by DNA sequencing. We conclude that the AID TB Resistance line probe assay is an accurate tool for the rapid detection of resistance mutations in cultured isolates and in smear-positive clinical specimens.