Improved real-time PCR for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis clinical isolates

In this study we designed two pairs of probes for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis with real-time PCR procedures. One pair of probes spans the region between codon 510 and 528 of the rpoB gene, and the other one screens for mutation at the regulatory region of the inhA gene. We have evaluated these probes in combination with two other pairs of probes previously described to detect mutations in 20 susceptible and 53 unique resistant M. tuberculosis clinical isolates. We were able to detect nine different mutations affecting five codons of the rpoB gene, two different mutations at codon 315 of the katG gene and a nucleotide substitution (C209T) in the regulatory region of the inhA gene within two hours turnaround.     

耐多药结核分枝杆菌耐药相关基因突变特征分析

目的了解中国耐多药结核分枝杆菌耐药相关基因的分子特征。方法对138株耐多药结核分枝杆菌和45株敏感菌的耐药相关基因inhA、katG和oxyR-ahpC间隔区(异烟肼)、rpob(利福平)、gyrA(氧氟沙星)和rrs(卡那霉素)进行序列测定,分析其基因突变特点。结果 138株耐多药结核分枝杆菌中,14.4%的菌株inhA基因发生突变,72.5%菌株的katG基因发生突变,15.9%菌株的oxyR-ahpC基因发生突变,同时考虑这3种基因,异烟肼耐药相关基因突变检出率可达90.6%;94.2%菌株的rpoB基因发生突变,74.5%菌株的gyrA基因发生突变,61.1%菌株的rrs基因发生突变,主要的突变位点为katG315(66.7%),inhA-15(9.4%),oxyR-ahpC-10(5.1%),rpoB516(13.8%),526(26.1%)和531(49.3%),gyrA90(21.6%)和94(51%),rrs1401(61.1%)。结论我国耐多药结核菌异烟肼、利福平、氧氟沙星和卡那霉素耐药相关基因最常见突变为katG315、inhA-15,rpoB531、526和516,gyrA94和90,rrs1401。     

Characterization of mutations conferring extensive drug resistance to Mycobacterium tuberculosis isolates in Pakistan

The increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosis in high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosis strains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the "hot-spot" regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrs genes in 50 XDR M. tuberculosis strains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosis strains would be undetected if only these common hot-spot regions were tested. The frequencies of resistance-conferring mutations were found to be comparable among all XDR M. tuberculosis strain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDR M. tuberculosis strains.     

Molecular detection of mutations associated with first- and second-line drug resistance compared with conventional drug susceptibility testing of Mycobacterium tuberculosis

The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.     

耐药结核分枝杆菌基因突变分析

目的 探讨结核分枝杆菌耐药表型与基因突变位点之间的相互关系.方法 采用序列特异性引物分别扩增92株结核分枝杆菌利福平耐药基因rpoB,异烟肼耐药基因katG、inhA、ahpC,链霉素耐药基因rrs、rpsL,乙胺丁醇耐药基因embB及喹诺酮耐药基因gyrA,SSCP筛选出突变序列,DNA测序分析突变性质.结果 59株利福平耐药株rpoB基因突变检出率94.9%(56/59),以Ser450Trp突变最多;90株异烟肼耐药株中,katG基因突变检出率38.9%(35/90),以Ser315Thr最多,3株检出inhA基因突变,ahpC基因无突变检出;34株喹诺酮耐药株中gyrA基因突变检出率82.4%(28/34),主要为Asp94Gly,其次为Ala90Val;31株链霉素耐药株中,15株检出rrs突变,最常见为A514C和A1041G,10株发生rpsL Lys88Arg突变,总的链霉素基因突变检出率为77.4%(24/31);31株乙胺丁醇耐药株中embB 基因突变检出率19.4%(6/31),主要为Met306Val.结论 耐药结核分枝杆菌耐药情况较为严重,以DNA测序为基础的基因突变分析能快速有效地检测结核分枝杆菌的rpoB、katG、gyrA、rrs、rpsL、embB 等耐药分子标识,显示了西安地区耐药性结核分枝杆菌的突变特点,为结核病的临床诊断和合理用药提供了实验依据.     

应用多重PCR-单链构象多态性分析同时检测耐异烟肼和利福平的结核分枝杆菌

目的 探讨应用多重PCR-单链构象多态性分析(multiplexpulymerase chain reaction-single strand conformation polymorphism,multi-PCR-SSCP)方法快速、特异地同时快速检测结核分枝杆菌对异烟肼和利福平耐药性的效能.方法 根据结核分枝杆菌的inhA序列、katG序列、rpoB序列,分别设计出3对特异性寡聚核苷酸引物.采用multi-PCR-SSCP技术,一次性检出耐异烟肼和利福平的结核分枝杆菌.新方法的有效性通过116株临床分离株(70株耐异烟肼,66株耐利福平)的验证.结果 名 Multi-PCR-SSCP方法检测临床分离株基因突变的有效性,以细菌培养和药敏试验结果为金标准.116株临床分离株和H37Rv标准株中除了4株katG缺失突变,其余菌株3个基因katG、inhA和rpoB在单基因PCR中都扩增成功.与H37Rv标准株相比,46株katG基因突变,14株inhA基因突变,58株rpoB基因突变.38株katG和rpoB,4株inhA和rpoB,4株inhA和katG同时突变,还有2株3个基因都有突变.multi-PCR-SSCP对于耐异烟肼和利福平的结核分枝杆菌检出的敏感度分别为80%、82%,特异度分别为100%和92%.结论 multi-PCR-SSCP方法敏感、特异,能同时快速有效地检测耐多药结核分枝杆菌,有望成为临床指导用药的好方法,为深入研究耐药基凶检测奠定了良好的基础.     

Molecular analysis of isoniazid-resistant Mycobacterium tuberculosis isolates from England and Wales reveals the phylogenetic significance of the ahpC -46A polymorphism

The present study investigated the prevalence and diagnostic potential of the most commonly reported mutations associated with isoniazid resistance, katG 315Thr, katG 315Asn, inhA -15T, inhA -8A, and the oxyR-ahpC intergenic region, in a population sample of 202 isoniazid-resistant Mycobacterium tuberculosis isolates and 176 randomly selected fully sensitive isolates from England and Wales identified by using a directed oligonucleotide array and limited DNA sequencing. The strains were recovered from patients originating from 29 countries; 41 isolates were multidrug resistant. Mutations affecting katG 315, the inhA promoter, and the oxyR-ahpC intergenic region were found in 62.7, 21.9, and 30% of 169 genotypically distinct isoniazid-resistant isolates, respectively, whereas they were found in 0, 0, and 8% of susceptible strains, respectively. The frequency of mutation at each locus was unrelated to the resistance profile or previous antituberculous drug therapy. The commonest mutation in the oxyR-ahpC intergenic region, ahpC -46A, was present in 23.7% of isoniazid-resistant isolates and 7.5% of susceptible isolates. This proved to be a phylogenetic marker for a subgroup of M. tuberculosis strains originating on the Indian subcontinent, which shared IS6110-based restriction fragment length polymorphism and spoligotype features with the Delhi strain and Central Asian strain CAS1; and this marker is strongly associated with isoniazid resistance and the katG 315Thr mutation. In total, 82.8% of unrelated isoniazid-resistant isolates could be identified by analysis of just two loci: katG 315 and the inhA promoter. Analysis of the oxyR-ahpC intergenic region, although phylogenetically interesting, does not contribute significantly to further identification of isoniazid-resistant isolates.     

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolated in Nepal

Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.     

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.     

Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong

OBJECTIVES: To characterize 250 drug-resistant Mycobacterium tuberculosis (MTB) isolates in Hong Kong with respect to their drug susceptibility phenotypes to five common anti-tuberculosis drugs (ofloxacin, rifampicin, ethambutol, isoniazid and pyrazinamide) and the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes (gyrA, rpoB, embB, katG, inhA, ahpC and pncA). METHODS: The MIC values of the aforementioned anti-tuberculosis drugs were determined for each of the 250 drug-resistant MTB clinical isolates by the absolute concentration method. Genetic mutations in the corresponding resistance genes in these MTB isolates were identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer conformation analysis (SSCP/MPAC), followed by DNA sequencing of the purified PCR products. RESULTS: Resistance to four or five drugs was commonly observed in these MTB isolates; such phenotypes accounted for over 34% of the 250 isolates. The most frequently observed phenotypes were those involving both rifampicin and isoniazid, with or without additional resistance to the other drugs. A total of 102 novel mutations, which accounted for 80% of all mutation types detected in the 7 resistance genes, were recovered. Correlation between phenotypic and mutational data showed that genetic changes in the gyrA, rpoB and katG genes were more consistently associated with a significant resistance phenotype. Despite this, however, a considerable proportion of resistant MTB isolates were found to harbour no detectable mutations in the corresponding gene loci. CONCLUSIONS: These findings expand the spectrum of potential resistance-related mutations in MTB clinical isolates and help consolidate the framework for the development of molecular methods for delineating the drug susceptibility profiles of MTB isolates in clinical laboratories.     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis isolates collected in Australia

Elucidation of the molecular basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has led to the development of different genotypic approaches for the rapid detection of INH resistance in clinical isolates. Mutations in katG, in particular the S315T substitution, are responsible for INH resistance in a large proportion of tuberculosis cases. However, the frequency of the katG S315T substitution varies with population samples. In this study, 52 epidemiologically unrelated clinical INH-resistant M. tuberculosis isolates collected in Australia were screened for mutations at katG codon 315 and the fabG1-inhA regulatory region. Importantly, 52 INH-sensitive isolates, selected to reflect the geographic and genotypic diversity of the isolates, were also included for comparison. The katG S315T substitution and fabG1-inhA -15 C-to-T mutation were identified in 34 and 13 of the 52 INH-resistant isolates, respectively, and none of the INH-sensitive isolates. Three novel katG mutations, D117A, M257I, and G491C, were identified in three INH-resistant strains with a wild-type katG codon 315, fabG1-inhA regulatory region, and inhA structural gene. When analyzed for possible associations between resistance mechanisms, resistance phenotype, and genotypic groups, it was found that neither the katG S315T nor fabG1-inhA -15 C-to-T mutation clustered with any one genotypic group, but that the -15 C-to-T substitution was associated with isolates with intermediate INH resistance and isolates coresistant to ethionamide. In total, 90.4% of unrelated INH-resistant isolates could be identified by analysis of just two loci: katG315 and the fabG1-inhA regulatory region.     

Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis

Rifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance of Mycobacterium leprae and Mycobacterium tuberculosis results from a mutation in the rpoB gene, encoding the β subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for rifampin resistance using clinical isolates present some limitations. Each clinical isolate has its own genetic variations in some loci other than rpoB, which might affect rifampin susceptibility. For this study, we constructed recombinant strains of Mycobacterium smegmatis carrying the M. leprae or M. tuberculosis rpoB gene with or without mutation and disrupted their own rpoB genes on the chromosome. The rifampin and rifabutin susceptibilities of the recombinant bacteria were measured to examine the influence of the mutations. The results confirmed that several mutations detected in clinical isolates of these two pathogenic mycobacteria can confer rifampin resistance, but they also suggested that some mutations detected in M. leprae isolates or rifampin-resistant M. tuberculosis isolates are not involved in rifampin resistance.     

Prevalence of and molecular basis for tuberculosis drug resistance in the Republic of Georgia: validation of a QIAplex system for detection of drug resistance-related mutations

We developed a QIAplex system for the simultaneous detection of 24 Mycobacterium tuberculosis gene mutations responsible for resistance to isoniazid (INH), rifampin (RIF), streptomycin (STM), and ethambutol (EMB) in 196 M. tuberculosis isolates recovered in the Republic of Georgia. In comparison to phenotypic susceptibility tests, the QIAplex showed sensitivity and specificity of 85.4% and 96.1% for INH, 94.4% and 99.4% for RIF, 69.6% and 99.2% for STM, 50.0% and 98.8% for EBM, and 86.7% and 100.0% for multidrug resistance, respectively. The dominant resistance mutations revealed were a mutation in katG resulting in S315T (katG S315T), rpsL K43R, and rpoB S531L. Mutations katG S315G and S315T and rpoB S531L were detected with higher frequencies in pretreated patients than in naive patients (P < 0.05). Simultaneous detection of 24 common drug resistance-related mutations provides a molecular tool for studying and monitoring M. tuberculosis resistance mechanism and epidemiology.     

Characterization of the katG and inhA genes of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis.

Resistance to isoniazid in Mycobacterium tuberculosis has been associated with mutations in genes encoding the mycobacterial catalase-peroxidase (katG) and the InhA protein (inhA). Among the 26 isoniazid-resistant clinical isolates evaluated in this study, mutations in putative inhA regulatory sequences were identified in 2 catalase-positive isolates, katG gene alterations were detected in 20 strains, and 4 isolates had wild-type katG and inhA genes. Mutations in the katG gene were detected in all 11 catalase-negative isolates: one frameshift insertion, two partial gene deletions, and nine different missense mutations were identified. An arginine-to-leucine substitution at position 463 was detected in nine catalase-positive isolates. However, site-directed mutagenesis experiments demonstrated that the presence of a leucine at codon 463 did not alter the activity of the M. tuberculosis catalase-peroxidase and did not affect the capacity of this enzyme to restore isoniazid susceptibility to isoniazid-resistant, KatG-defective Mycobacterium smegmatis BH1 cells. These studies further support the association between katG and inhA gene mutations and isoniazid resistance in M. tuberculosis, while also suggesting that other undefined mechanisms of isoniazid resistance exist.     

Molecular basis of rifampin and isoniazid resistance in Mycobacterium bovis strains isolated in Sardinia, Italy

Fourteen of 22 (68%) Mycobacterium bovis strains isolated from cattle in Sardinia were found to be resistant to rifampin and isoniazid. Analysis of the rpoB and the katG, oxyR-ahpC, and inhA gene regions of these strains was performed in order to investigate the molecular basis of rifampin and isoniazid resistance, respectively. The most frequent mutation, encountered in 6 of 10 strains (60%), was in the rpoB gene; it occurred, at codon position 521 and resulted in leucine changed to proline. This suggests that codon 521 may be important for the development of rifampin resistance in M. bovis. Resistance to isoniazid is associated in Mycobacterium tuberculosis with a variety of mutations affecting one or more genes. Our results confirm the difficulty of interpreting the sequence variations observed in clinical strains of M. bovis. M. bovis strains isolated from the same geographic area showed similar mutations within the genes responsible for rifampin and isoniazid resistance. Our results represent the first study to elucidate the molecular genetic basis of drug resistance in M. bovis isolated from cattle.     

基因芯片技术在结核分支杆菌耐药突变基因检测中的应用

目的通过基因芯片检测系统，快速检测临床样品中结核分支杆菌耐药突变情况。方法根据结核分支杆菌标准株H37Rv序列，设计了覆盖rpoB、katG，inhA基因突变区的系列寡核苷酸探针，制作膜芯片，检测临床样品中结核分支杆菌基因突变情况，以此判断耐药结果。结果在305例临床病例中，共检出阳性病例125例，其中阳性敏感病例64例，阳性突变病例61例，阳性率为40．98％，在125例阳性样品中，共发现有8种突变类型，其中10例531L，占7．94％，19例315M，占阳性样品中总数的15．08％。结论PCR与膜芯片杂交技术可临床检测结核分支杆菌对利福平和异烟肼的耐药性，并具有快速、简便、敏感的特点。     

Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis

Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter -15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter -15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.     

Molecular characteristics of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis isolates from the Russian Federation

OBJECTIVES: Three Mycobacterium tuberculosis genetic loci--rpoB and katG genes and the fabG1(mabA)-inhA operon promoter region--were studied to reveal the mutations associated with rifampicin and isoniazid resistance. METHODS: Four hundred and twelve isolates of M. tuberculosis from different regions of the Russian Federation were collected during 1997-2005. A matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based minisequencing method was used for the detection of mutations. RESULTS: Thirteen different variants of single mutations in codons 533, 531, 526, 516, 513 and 511 of the rifampicin resistance-determining region of the rpoB gene as well as the TTG insertion in the 514a position were found among the rifampicin-resistant isolates. Single nucleotide substitutions in codons 531, 526 and 516 (64.8%, 10.3% and 7.7%, respectively) were the most prevalent mutations. Codon 526 was shown to be the most variable of all. No mutations were detected in rpoB genes for 29 (10.7%) of the rifampicin-resistant isolates. 76.9% of the isoniazid-resistant isolates carried single mutations in codon 315 of the katG gene. For another 12.9% of them, double mutations in the katG gene and the fabG1(mabA)-inhA promoter region were revealed. No mutations were detected in 8.2% of the isoniazid-resistant isolates. CONCLUSIONS: Molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. The new MALDI-TOF MS-based method may be used for rapid and accurate monitoring of the spread of drug resistance.     

Molecular analysis of isoniazid resistance in Mycobacterium tuberculosis isolates recovered from South Korea

The katG, inhA and ahpC genes, in 71 isoniazid (INH)-resistant and 26 INH-susceptible Mycobacterium tuberculosis isolates, from South Korea were examined by sequencing and MspI restriction enzyme analysis. Mutations in the katG 315 alone, katG 315 plus inhA, katG 315 plus ahpC, katG 309 alone, katG 309 plus inhA, inhA alone, and ahpC alone, were detected in 54.9, 2.8, 1.4, 1.4, 1.4, 19.7, and 5.6% of the 71 INH-resistant isolates, respectively. There was no statistically significant difference (p > 0.05) in the frequencies of these mutations for the INH-monoresistant compared with the multidrug-resistant isolates. Mutations in the katG codon 315 were associated with the high-level of INH resistance (MIC, >1 microg/ml), whereas the mutation in the inhA promoter region was associated with the low-level of INH resistance (MIC, >0.2 to 1 microg/ml). The previously undescribed GGT-->GAT (Gly-->Asp) mutation in the katG codon 309 was found in two rifampin, including-multidrug-resistant isolates, but we cannot assess if this is predictive of INH resistance. The sensitivity and specificity of molecular analysis of the katG codon 315 and/or the inhA promoter region were 80.3 and 100%, respectively. Therefore, mutations in these regions are highly predictive of INH resistance in South Korea.     

Evaluation of the CombiChip Mycobacteria Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis

The CombiChip Mycobacteriatrade mark Drug-Resistance Detection DNA chip, recently developed by GeneIn (Pusan, South Korea), is an oligonucleotide microchip coupled with polymerase chain reaction for the detection of mutations associated with resistance to isoniazid (INH) and rifampin (RIF). This oligonucleotide chip was compared with DNA sequencing and phenotypic drug susceptibility testing with 69 INH- and/or RIF-resistant and 27 all tested drug-susceptible Mycobacterium tuberculosis isolates. Two selected codons (the katG codon 315 and inhA15) allowed identification of 84.1% of INH-resistant isolates and 100% of RIF resistance were detected by screening for 7 codons: rpoB511, rpoB513, rpoB516, rpoB522, rpoB526, rpoB531, and rpoB533. The overall specificity of this oligonucleotide chip for detecting INH and RIF resistance were 100 and 95.3%, respectively. This level of sensitivity and specificity is concordant with that from the determination of M. tuberculosis drug resistance by DNA sequencing. This oligonucleotide chip is a rapid and reliable genotypic method capable of detecting multiple mutations associated with INH and RIF resistance simultaneously in a single microchip slide.