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.     

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.     

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.     

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.     

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).     

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.     

Detecting mutation pattern of drug-resistant Mycobacterium tuberculosis isolates in Himachal Pradesh using GenoType® MTBDRplus assay

Context: Tuberculosis (TB) is a major public health problem in India and a principal cause of death in adults, especially among the economically productive age group. India accounts for one-fifth of the global burden of TB. It is estimated that about 40% of Indian population is infected with TB bacillus. The GenoType® MTBDRplus molecular method allows rapid diagnosis of the clinical samples and detection of the most common mutations in the genes associated with rifampicin (R) and isoniazid (H) resistance. Aims: To study the drug resistance and mutational patterns in multidrug-resistant (MDR) suspects clinical strains using GenoType® MTBDRplus assay. Subjects and Methods: A total of 770 sputum samples of the MDR-TB suspects were included in this study, which were received at Intermediate Reference Laboratory, Government TB Sanatorium, Dharampur, Solan, Himachal Pradesh from the Designated Microscopy Centres of Himachal Pradesh for the culture and susceptibility testing. All the 521 Mycobacterium tuberculosis complex (MTBC) strains were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay to detect molecular resistance pattern to first line anti-tubercular drugs (isoniazid and rifampicin). Results: Of 770 samples, 556 (72.20%) were from male and 214 (27.80%) were from female. Among the 521 MTBC strains, 19.76% were found to be MDR and mono-resistance to isoniazid and rifampicin was detected in 8.63% and 6.14% strains respectively. About 74.81%, 76.35% and 5.40% strains harboured known mutation in rpoB, katG and inhA genes respectively. Conclusions: In rpoB gene, the most common mutation is associated with S531 L region. The GenoType® MTBDRplus assay is a rapid test for the detection of the most common mutations in MDR-TB strains. In our study, unknown rpoB gene mutations were found in 25.18% strains that may further be detected by gene sequencing.     

Multidrug-resistant tuberculosis: rapid detection of resistance to rifampin and high or low levels of isoniazid in clinical specimens and isolates

The aim of the present study was to evaluate a new improved multiplex polymerase chain reaction (PCR) hybridisation assay to detect multidrug-resistant tuberculosis. The assay, developed to detect rifampin (rpoB) and isoniazid (katG) gene mutations causing Mycobacterium tuberculosis resistance, was recently extended to include inhA gene mutations that code for low-level isoniazid resistance. Interpretable results were obtained in 115 isolates and in all smear-positive clinical specimens. Rifampin resistance was correctly identified in all specimens and in 20 of 21 (95%) multidrug-resistant isolates compared to BACTEC 460TB. Isoniazid resistance correlated in 18 of 22 (82%) specimens, in 31 of 31 (100%) high-level and 24 of 28 (86%) low-level isoniazid-resistant isolates. The assay was rapid, easy to perform and directly applicable in smear-positive specimens. We predict that the assay may be a useful tool to combat and prevent new cases of multi- and extensively drug-resistant tuberculosis.     

Rolling circle amplification and multiplex allele-specific PCR for rapid detection of katG and inhA gene mutations in Mycobacterium tuberculosis

The aim of the study was to compare a novel, rolling circle amplification (RCA) assay for detection of common isoniazid (INH) resistance mutations in Mycobacterium tuberculosis with a multiplex allele-specific PCR (MAS-PCR) and sequencing of katG and the fabG1-inhA promoter region. One or more mutations were identified by RCA, MAS-PCR, and sequencing in 21 (68%), 22 (71%), and 23 (74%), respectively, of 31 epidemiologically unrelated INH-resistant isolates, and in none of 8 INH-susceptible isolates. The RCA assay is a rapid, inexpensive, and practical screening method for INH resistance in M. tuberculosis in countries with high prevalence of INH resistance.     

High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin Resistance in Mycobacterium tuberculosis: a Meta-Analysis

A rapid, simple, accurate, and affordable method for the detection of drug-resistant tuberculosis is very critical for the selection of antimicrobial therapy and management of patient treatment. High-resolution melting curve analysis has been used for the detection of rifampin resistance in Mycobacterium tuberculosis and has shown promise. We did a systematic review and meta-analysis of published studies to evaluate the accuracy of high-resolution melting curve analysis for the detection of rifampin resistance in clinical M. tuberculosis isolates. We searched the PubMed, BIOSIS Previews, and Web of Science databases to identify studies and included them according to predetermined criteria. We used the DerSimonian-Laird random-effects model to calculate pooled measures and applied Moses'' constant for linear models to fit the summary receiver operating characteristic curve. According to the selection criteria, most of the identified studies were excluded, and only seven studies were included in the final analysis. The overall sensitivity of the high-resolution melting curve analysis was 94% (95% confidence interval [CI], 92% to 96%), and the overall specificity was very high at 99% (95% CI, 98% to 100%). The values for the pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 63.39 (95% CI, 30.21 to 133.00), 0.06 (95% CI, 0.04 to 0.09), and 892.70 (95% CI, 385.50 to 2,067.24), respectively. There was no significant heterogeneity across all included studies for the measurements we evaluated. The summary receiver operating characteristic curve for the same data shows an area of 0.99 and a Q* value of 0.97. High-resolution melting curve analysis has high sensitivity and specificity for the detection of rifampin resistance in clinical M. tuberculosis isolates. This method might be a good alternative to conventional drug susceptibility tests in clinical practice.     

Whole-genome sequencing for surveillance of tuberculosis drug resistance and determination of resistance level in China

ObjectivesPhenotypic drug susceptibility testing for prediction of tuberculosis (TB) drug resistance is slow and unreliable, limiting individualized therapy and monitoring of national TB data. Our study evaluated whole-genome sequencing (WGS) for its predictive accuracy, use in TB drug-resistance surveillance and ability to quantify the effects of resistance-associated mutations on MICs of anti-TB drugs.MethodsWe used WGS to measure the susceptibility of 4880 isolates to ten anti-TB drugs; for pyrazinamide, we used BACTEC MGIT 960. We determined the accuracy of WGS by comparing the prevalence of drug resistance, measured by WGS, with the true prevalence, determined by phenotypic susceptibility testing. We used the Student–Newman–Keuls test to confirm MIC differences of mutations.ResultsResistance to isoniazid, rifampin and ethambutol was highly accurately predicted with at least 92.92% (95% confidence interval [CI], 88.19–97.65) sensitivity, resistance to pyrazinamide with 50.52% (95% CI, 40.57–60.47) sensitivity, and resistance to six second-line drugs with 85.05% (95% CI, 80.27–89.83) to 96.01% (95% CI, 93.89–98.13) sensitivity. The rpoB S450L, katG S315T and gyrA D94G mutations always confer high-level resistance, while rpoB L430P, rpoB L452P, fabG1 C-15T and embB G406S often confer low-level resistance or sub-epidemiological cutoff (ECOFF) MIC elevation.ConclusionWGS can predict phenotypic susceptibility with high accuracy and could be a valuable tool for drug-resistance surveillance and allow the detection of drug-resistance level; It can be an important approach in TB drug-resistance surveillance and for determining therapeutic schemes.     

Utility of GenoType MTBDRplus assay in rapid diagnosis of multidrug resistant tuberculosis at a tertiary care centre in India

Purpose: Molecular methods which allow rapid detection of tuberculosis as well as drug resistance directly from clinical samples have become the most popular diagnostic methodology with the emergence of multidrug resistant tuberculosis. The aim of the present study was to evaluate the performance of a line probe assay, GenoType MTBDRplus for the rapid detection of Mycobacterium tuberculosis and mutations causing rifampicin and INH resistance directly in smear positive pulmonary specimens and also in M. tuberculosis isolates grown from various clinical specimens. Materials and Methods: The MTBDRplus assay was done directly on 37 smear positive pulmonary specimens and also on 69 M. tuberculosis isolates obtained by rapid automated culture using Bact/Alert 3D. The results were compared with phenotypic drug susceptibility testing (1% proportion method) using Bact/Alert 3D. Results: The sensitivity and specificity for detection of resistance to rifampicin was 100% and 97.3%, and to INH was 91.9% and 98.4%, respectively, in comparison with the phenotypic drug susceptibility testing. Conclusion: MTBDRplus assay had good sensitivity and specificity with turn around time of less than 48 hours. It may be a useful tool for rapid detection of multidrug resistant tuberculosis at a tertiary care centre.     

Visual Detection of rpoB Mutations in Rifampin-Resistant Mycobacterium tuberculosis Strains by Use of an Asymmetrically Split Peroxidase DNAzyme

Multidrug-resistant Mycobacterium tuberculosis is resistant to two first-line antituberculosis drugs, isoniazid and rifampin, resulting in the relapse of tuberculosis. M. tuberculosis grows very slowly, and thus traditional examination methods take time to test its drug resistance and cannot meet clinical needs. The use of a DNA probe makes it possible to test rifampin resistance. We developed an asymmetrical split-assembly DNA peroxidase assay to detect drug-resistant mutation of rifampin-resistant M. tuberculosis in the rpoB gene rapidly and visibly. A new strategy was also designed to eliminate the adverse effects caused by the complicated secondary structure of the target DNA and to improve the efficiency of the probes. This detection system consists of five group detections, covers rifampin-resistant determination region of the rpoB gene, and tests 40 kinds of mutations, including the most common mutations at codons 531 and 526. Every group detection or individual mutant allele detection can distinguish corresponding mutant DNA sequences from the wild-type DNA sequences.     

Prospective evaluation of pyrosequencing for the rapid detection of isoniazid and rifampin resistance in clinical <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates

A pyrosequencing-based method for the rapid detection of isoniazid (INH) and rifampin (RIF) resistance in Mycobacterium tuberculosis was evaluated in clinical practice. The method can detect the INH resistance-causing katG315 mutation, and all mutations in the RIF resistance-determining rpoB core region, in less than 6 h from cultured isolates. The method was first validated with 42 isolates, and was subsequently prospectively evaluated with 91 isolates, including clinical isolates and external quality control assessment strains, over a period of 2.5 years. The pyrosequencing results of clinical isolates were available, on average, 19 days earlier (median 19 days; range 3–43 days) than conventional susceptibility testing results. The composite data showed that the sensitivity of pyrosequencing for detecting resistance correctly was 66.7% for INH and 97.4% for RIF. The specificity of pyrosequencing was 100% for both drugs. Acceptable sensitivity for detecting resistance and the rapidness of pyrosequencing make it a valuable tool in the clinical setting.     

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.     

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.     

Detection of Ofloxacin Resistance by Nitrate Reductase Assay in Mycobacterium tuberculosis Isolates from Extrapulmonary Tuberculosis

Context: Increased use of fluoroquinolones to treat community-acquired infections has led to the decreased susceptibility to Mycobacterium tuberculosis. There is a paucity of data on ofloxacin (OFX) resistance detection by nitrate reductase assay (NRA). Hence, the present study was carried out to find the efficacy of NRA for detection of OFX resistance in M. tuberculosis isolated from extrapulmonary tuberculosis (EPTB) cases. Aims: (1) To compare sensitivity, specificity and median time required to obtain results by NRA with economic variant proportion method (PM) for detection of OFX resistance.(2) To determine the extent of OFX resistance in clinical isolates of M. tuberculosis. Settings and Design: Seventy-three M. tuberculosis isolates from cases of EPTB were subjected to economic variant of PM for isoniazid, rifampicin and OFX. NRA was done for detection of OFX resistance. Subjects and Methods: Seventy-three isolates from clinical samples of suspected EPTB received in the Department of Microbiology were included in the study. Drug susceptibility test was performed on Lowenstein– Jensen medium with and without drugs. Statistical Analysis Used: Of turnaround time was done by Mann–Whitney test on SPSS (version 19, released in 2010, IBM Corp, Armonk NY), P < 0.05. Results: OFX resistance was seen in nine isolates. The sensitivity and specificity of OFX resistance by NRA was 100% and 96.87%, respectively. Median time required to obtain results by NRA was 10 days as compared to 28 days by PM. Conclusions: NRA is a specific and sensitive method for detection of OFX resistance in resource-restricted settings.     

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.     

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis: its utility in resource poor settings

Purpose: To compare the rapid colorimetric nitrate reductase based antibiotic susceptibility (CONRAS) test performed on Mycobacterium tuberculosis isolates with the conventional method i.e., the proportion method. Methods: One hundred clinical isolates of M. tuberculosis were tested for susceptibility to isoniazid (INH) and rifampicin (RIF) by the conventional proportion method and CONRAS in Middlebrook 7H9 liquid medium enriched with growth supplements (MB7H9S). Results: The performance of the CONRAS test was evaluated using proportion method as the gold standard. The sensitivity (ability to detect true drug resistance) and specificity (ability to detect true drug susceptibility) of the CONRAS test to INH was 93.75 and 98.52% and for RIF it was 96.10 and 100% respectively. The mean time for reporting was 6.3 days and the test showed excellent reproducibility. The kappa (κ) value for INH was 0.92 and for RIF was 0.99, indicating excellent agreement between the two methods. Conclusions: CONRAS test is a rapid and reliable method of drug susceptibility for M. tuberculosis.     

High-Resolution Melting Curve Analysis for Rapid Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Clinical Isolates

We evaluated high-resolution melting (HRM) curve analysis as a tool for detecting rifampin (RIF) and isoniazid (INH) resistance in Mycobacterium tuberculosis in an accurate, affordable, and rapid manner. Two hundred seventeen M. tuberculosis clinical isolates of known resistance phenotype were used. Twenty-nine known rpoB mutant DNAs, including rare mutations, were also included. Four pairs of primers were designed: rpoB-F/R (for codons 516 to 539 of rpoB), rpoB-516F/R (for codons 508 to 536 of rpoB), katG-F/R (for the codon 315 region of katG), and inhA-F/R (for the nucleotide substitution of C to T at position −15 of inhA). An HRM curve was generated for each isolate after real-time PCR differentiated the mutant from the wild-type strains. DNA sequencing of the target regions was performed to confirm the results of the HRM curve analysis. All but one of the 73 RIF-resistant (RIF-R) strains and all 124 RIF-susceptible (RIF-S) isolates were correctly identified by HRM curve analysis of rpoB. Twenty-seven of 29 known rpoB mutants were detected. In HRM curve analysis of katG and inhA, 90 INH-R strains that harbored katG or inhA mutations, or both, and all INH-S strains were correctly identified. Ten phenotypically INH-R strains not harboring katG or inhA mutations were not detected. The HRM curve analysis will be a useful method for detection of RIF and INH resistance in M. tuberculosis in a rapid, accurate, simple, and cost-effective manner.The rates of mortality and morbidity from tuberculosis (TB) remain high, despite intense worldwide efforts. One of the major factors sustaining the current TB epidemic is the increasing drug resistance of Mycobacterium tuberculosis strains (2). In the early 1990s, multidrug-resistant (MDR) TB cases that were resistant to at least rifampin (RIF) plus isoniazid (INH) arose (6). When the frequency and distribution of extensively drug-resistant (XDR) TB cases were assessed in 2004 by the U.S. Centers for Disease Control and Prevention and the World Health Organization, several cases of drug-resistant tuberculosis consistent with an XDR phenotype were found (7). This study revealed that 20% of the isolates met the MDR criteria; 2% of those were classifiable as XDR; and 4%, 15%, and 19% of the XDR TB cases were from the United States, South Korea, and Latvia, respectively (7). Thus, it is crucial that rapid drug susceptibility tests be developed to prevent the spread of MDR and XDR TB.Although drug susceptibility testing (DST) is a prerequisite for accurate results, such testing requires much time and labor (3). Therefore, several molecular techniques have been applied to detect mutations related to drug resistance (5, 10). Resistance to RIF and INH, the mainstays of antituberculosis treatment, is mainly attributable to mutations in genes encoding the drug target or drug-converting enzymes (8). Early studies demonstrated that 95% of the resistance to RIF is associated with mutation of the RIF resistance-determining region of rpoB, whereas mutations in katG and the regulatory zone of inhA are most frequently associated with INH resistance (11).The oligonucleotide chip method and real-time PCR have been used for detection of drug-resistant M. tuberculosis (17, 21, 27, 29). A novel method of high-resolution melting (HRM) curve analysis is an accurate and simple technique for analyzing the genotype without the need for specific probes. The dye LC Green, SYTO9, or Eva Green saturates amplified DNA, unlike SYBR green dye, during homogeneous melting curve analysis. Also, HRM curve analysis generates a difference plot curve, which analyzes nucleic acid sequences with high accuracy. Application of genotyping by HRM curve analysis has followed (13, 19). The aim of the study described here was to develop a useful molecular tool for the identification of drug resistance in M. tuberculosis in an accurate, rapid, and cost-effective manner.