Evaluation of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

A novel PCR-based reverse hybridization method Genotype MTBDR assay (Hain Lifescience GmbH, Nehren, Germany) was evaluated for rapid detection of rifampin (RIF) and isoniazid (INH) resistance in Turkish Mycobacterium tuberculosis isolates. The Genotype MTBDR assay is designed to detect mutations within the 81-bp hotspot region of rpoB and mutations at katG codon 315. A total of 41 RIF-resistant M. tuberculosis isolates with rpoB mutations that were previously tested by the INNO-LiPA Rif.TB kit and also characterized by DNA sequencing were included in the study. Thirty-seven of these isolates were also resistant to INH. RIF resistance was correctly identified in 39 of 41 isolates (95.1%) with the Genotype MTBDR assay probes specific for these mutations. One isolate with a Gln-490-His mutation and another one with a CGG insertion between codons 514 and 515 were identified as RIF sensitive by the Genotype MTBDR assay. While the INNO-LiPA Rif.TB kit was able to determine the CGG insertion between codons 514 and 515, the Gln-490-His mutation outside the 81-bp hotspot region was not detected by the INNO-LiPA Rif.TB kit. These isolates had MICs of >or=32 microg/ml for RIF. The Genotype MTBDR assay also correctly identified 27 of 37 INH-resistant isolates (73%) with mutations in katG codon 315. In conclusion, the Genotype MTBDR assay may be useful for the rapid diagnosis of the most common mutations found in multidrug-resistant M. tuberculosis strains. However, the test results should always be confirmed with phenotypic methods.     

Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance

We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and katG genes. One hundred thirteen M. tuberculosis isolates were tested. The nucleotide sequences of the katG and inhA genes and the mabA-inhA promoter region were also determined. The MTBDR assay detected 100% and 67% (n = 64) of the strains resistant to RIF and INH, respectively. Among the latter, 62 strains carried a Ser315Thr mutation in katG, 59 of them displaying a high level of resistance to INH. Two strains with a low level of INH resistance had a Ser315Asn mutation. No mutation was found by the MTBDR assay for 31 INH-resistant strains (33%), of which 24 showed a low level of resistance. By DNA sequencing, we found among them various mutations in the KatG protein for 7 strains, a C-->T mutation in position -15 of the mabA-inhA promoter in 17 strains, and a Ser94Ala mutation in InhA for 7 strains. In conclusion, the MTBDR assay, which fits easily in the workflow of a routine laboratory, enabled the detection of 100% of the RIF-resistant strains and 89% of the INH-resistant strains with a high level of resistance but only 17% of the strains characterized by a low level of INH resistance, indicating that the test can be used as a rapid method to detect in the same experiment the rifampin-resistant and the high-level isoniazid-resistant strains of M. tuberculosis.     

结核分枝杆菌临床分离株异烟肼耐药相关基因突变的分子特征

目的 阐明结核分枝杆菌异烟肼（INH）耐药相关基因突变特征.方法 对137株结核分枝杆菌临床分离株（耐异烟肼菌株87株,异烟肼敏感菌株90株）的9个结构基因furA、katG、inhA、kasA、Rv0340、iniB、iniA、iniC和efpA以及两个调控区oxyR-ahpC基因间隔区和mabA-inhA启动子进行DNA片段扩增及序列分析.结果 82株（94.3%）INH耐药分离株的katG基因存在突变,其中katGSer315Thr突变占优势（55.2%）.50株INH敏感的分离菌katG的463密码子没有突变.35株（40.2%）INH耐药的分离株katG的463有突变.87株INH耐药株中,20株（23.0%）的katG基因存在两重突变.13株（14.9%）分离菌inhA基因的启动子区存在突变,4.6%的分离菌有inhA结构基因突变,11.5%oxyR-ahpC基因间区存在突变.iniBAC区域和efpA中发现耐药性关联突变.结论 研究证实多个基因突变与异烟肼耐药之间的关系,并且为阐明结核分枝杆菌耐药机制提供线索.     

Molecular characterization of rifampin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Poland

A total of 105 rifampin (RMP)- and/or isoniazid (INH)-resistant strains of Mycobacterium tuberculosis isolated from different parts of Poland in 2000 were screened for mutations associated with resistance to these drugs by two molecular methods, namely sequence analysis and real-time PCR technology. Three loci associated with drug resistance were selected for characterization: they were rpoB (RMP), katG, and the regulatory region of inhA (INH). Nineteen different mutations were identified in 64 RMP-resistant strains, and five new alleles were described. The most common point mutations were in codons 531 (41%), 516 (16%), and 526 (9%) of the rpoB gene. Mutations were not found in two (3%) of the isolates. In the case of resistance to INH, six different mutations in the katG gene of 83 resistant strains were detected. Fifty-seven (69%) isolates exhibited nucleotide substitutions at codon 315. One strain harbored a mutation affecting codon 279 (Gly279Thr). Twelve of 26 INH-resistant strains with the wild-type codon 315 (14.5% of all strains tested) had the mutation -15C-->T in the regulatory region of inhA. A full correlation between the DNA sequence analysis and real-time PCR data was obtained. We conclude that the real-time PCR method is fast and reliable for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.     

Molecular characterization of isoniazid and rifampin resistance of Mycobacterium tuberculosis clinical isolates from Malatya, Turkey

Molecular characterization of drug resistance of Mycobacterium tuberculosis strains of different origins can generate information useful for developing molecular methods that are widely applicable for rapid drug resistance detection. Using DNA sequencing and allele-specific polymerase chain reaction (AS-PCR), we investigated genetic mutations associated with isoniazid (INH) and rifampin (RIF) resistance among 29 drug-resistant clinical isolates of M. tuberculosis collected from Malatya, Turkey, including 19 multi-drug-resistant (MDR) isolates. Point mutations were detected at codons 531, 516, 526, and 513 of the RNA polymerase beta- subunit gene (rpoB) in 10 (47.6%), five (23.8%), three (14.3%), and three (14.3%) of the 21 RIF-resistant isolates, respectively. Of the five isolates having mutations in codon 516, three also had mutations at codon 527; one had a concurrent mutation at codon 572. Mutations at codon 315 of the catalase-peroxidase-encoding gene (katG) were found in 17 (63.0%) of the 27 INH-resistant isolates. Interestingly, the katG codon 315 mutation was observed at a much higher frequency in MDR isolates than in INH-mono-resistant isolates ( approximately 79% vs. 25%). This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis clinical isolates from Eastern Turkey, and extended our knowledge of molecular basis of M. tuberculosis drug resistance.     

Detection of mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates from China

Nine structural genes (furA, katG, inhA, kasA, Rv0340, iniB, iniA, iniC, and efpA) and two regulatory regions (the oxyR-ahpC intergenic region and the promoter of mabA-inhA) in 87 isoniazid (INH)-monoresistant and 50 INH-susceptible Mycobacterium tuberculosis isolates collected from five provinces of China were analyzed by sequencing. Eighty-two (94.3%) INH-resistant isolates had mutations in the katG gene, with the katG Ser315Thr mutation predominant (55.2%). No mutation at codon 463 of katG was detected among the 50 INH-susceptible isolates with different IS6110 fingerprints. In addition, there were 35 (40.2%) INH-resistant isolates that had a mutation at codon 463 of katG. Of the INH-resistant strains, 20 (23.0%) isolates harbored double mutations at two separate loci of katG. Mutations in the inhA promoter region occurred in 13 (14.9%) isolates; 4.6% of the isolates had inhA structural gene mutations, and 11.5% harbored mutations in the oxyR-ahpC intergenic region. Drug resistance-associated mutations were detected in the iniBAC region and efpA.     

Frequency of mutations in rpoB and codons 315 and 463 of katG in rifampin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates from northeast Mexico

A total of 48 isoniazid (INH)- and rifampin (RIF)-resistant Mycobacterium tuberculosis isolates, 19 INH-resistant isolates, and 9 RIF-resistant isolates were randomly selected and tested for detecting mutations at codons 315 and 463 of katG by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and/or for detecting mutations at a 69-bp region of the rpoB gene by the INNO-LiPA Rif TB assay. Of the 67 INH-resistant isolates tested, 36 (53.7%) showed the mutation at codon 315 of katG; however, none of them showed the mutation at codon 463. The majority of the RIF-resistant samples analyzed (49 of 57, 86.0%) reacted positive with one of the four R-type probes. The R5-pattern (S531L mutation) was the most frequently observed (31 of 57, 54.4%), followed by R4a-pattern (H526Y mutation) 13 isolates (22.8%), R4b-pattern (H526D mutation) 4 isolates (7.0%), and R2-pattern (D516V mutation) 1 isolate (1.8%). Overall, there was agreement between the line probe kit and phenotypic RIF-susceptibility test for 56 (98.2%) of 57 RIF-resistant isolates tested. These results show that the mutation analysis at codon 315 of katG could be used as a screening assay prior to standard susceptibility testing, whereas mutations in the rpoB gene could be used successfully as genetic markers to rapidly detect RIF-resistant M. tuberculosis clinical isolates from northeast Mexico.     

Detection of multidrug resistance in Mycobacterium tuberculosis

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.     

Molecular characterization of multidrug- and extensively drug-resistant Mycobacterium tuberculosis strains in Jiangxi, China

In this study, a total of 77 multidrug-resistant and extensively drug-resistant (MDR and XDR, respectively) isolates of Mycobacterium tuberculosis were characterized among samples from patients living in Jiangxi province, China. The following two approaches were used: (i) genotyping all drug-resistant isolates by the 15-locus MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) method and identifying the Beijing family genotype using the RD105 deletion targeted multiplex PCR and (ii) determining the mutation profiles associated with the resistance to the first-line antituberculous drugs rifampin (RIF) and isoniazid (INH) and the second-line drugs ofloxacin (OFX), kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) with DNA sequencing. Six loci were examined: rpoB (for resistance to RIF), katG and mabA-inhA (INH), gyrA and gyrB (OFX), and rrs (KAN, AMK, and CAP). It is shown that the Beijing genotype was predominant (80.5%) among these strains and that the selected drug-resistant strains were genetically diverse, suggesting that they probably had independently acquired drug resistance. In comparison to the phenotypic data, the sensitivities for the detection of RIF, INH, OFX, and KAN/AMK/CAP resistance by DNA sequencing were 94.8, 80.5, 84.6, and 78.9%, respectively. The most prevalent mutations involved in RIF, INH, OFX, and KAN/AMK/CAP resistance were Ser531Leu in rpoB (44.2%), Ser315Thr in katG (55.8%) and C-15T in mabA-inhA (11.7%), Asp94Gly in gyrA (48.7%), and A1401G in rrs (73.7%), respectively. Five novel katG mutants (Trp191Stop, Thr271Pro, Trp328Phe, Leu546Pro, and Asp695Gly) and six new alleles (Ile569Val, Ile572Met, Phe584Ser, Val615Met, Asp626Glu, and Lys972Thr) in the rpoB gene were identified.     

Association of specific mutations in katG, rpoB, rpsL and rrs genes with spoligotypes of multidrug-resistant Mycobacterium tuberculosis isolates in Russia

Most multidrug-resistant (MDR) Mycobacterium tuberculosis isolates in Russia belong to the Beijing or Latino-American and Mediterranean (LAM) spoligotype families. The objective of this study was to investigate possible associations between genotype and the frequencies of mutations that confer drug resistance in a population that has two large families of circulating strains. Spoligotyping, IS6110 restriction fragment length polymorphism typing, and sequencing of the katG and rpoB genes, were performed for 217 consecutive MDR M. tuberculosis isolates from patients. The rpsL and rrs genes were also sequenced for selected streptomycin-resistant isolates. Of the 217 MDR isolates, 99 (46%) belonged to the LAM family, 92 (42%) to the Beijing family, 21 (10%) to the Haarlem family and four (2%) to the T family. There was one unique spoligotype. Mutations in the katG gene were identified in 207 (95%) isolates, all of which had mutations in codon 315. Mutations in the rpoB gene were identified in 200 (92%) isolates; 75% of LAM isolates carried a mutation in codon 516, whereas 71% of Beijing isolates carried a mutation in codon 531. In the 33 isolates resistant to streptomycin 50 mg/L, the 43AGG rpsL mutation was found in 27% of Haarlem, 75% of Beijing and 0% of LAM isolates, and rrs mutations were found in 17% (516C-->T) of Beijing and 100% (513A-->C) of LAM isolates. Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampicin or streptomycin in the Beijing and LAM families. The biological implications of this correlation remain to be explored.     

Mutation in katG315 is,possibly, a good prognostic marker for treatment with second-line drugs in multi-drug resistant tuberculosis: A preliminary study

The aim of this study was to explore baseline data, laboratory and molecular analyses to determine if any could serve as potential prognostic marker(s) for treatment response to second line tuberculosis regimens. Of a total number of 50 multi-drug resistant tuberculosis (MDR-TB) patients starting second-line drug MDR-TB treatment in Iraq, only 21 showed treatment adherence and thus, included in this study. Response to treatment was monitored for 11 months by sputum microscopy and culture. We explored baseline data, laboratory and molecular analyses to determine if any could serve as potential prognostic marker(s) for treatment response. Highly significant association (P = 0.019) was detected between mutations in katG315 codon and good response to second-line anti-TB drugs. Spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat confirmed that katG315-mutatnt isolates were genotypically unrelated. The katG315 mutation is a potential prognostic marker for treatment response to second-line anti-tuberculosis drugs. One possible explanation of our results is that the katG315-mutants are sensitive to bacterial killing by “oxidative killing.”     

The susceptibility of Mycobacterium tuberculosis to isoniazid and the Arg-->Leu mutation at codon 463 of katG are not associated

A mutation (CCG-->CTG [Arg-->Leu]) in codon 463 of katG (catalase peroxidase) of Mycobacterium tuberculosis has been found in isoniazid (INH)-resistant strains. A PCR restriction endonuclease analysis to detect this mutation was applied to 395 M. tuberculosis isolates from patients in The Netherlands. The proportion of isolates with a detectable mutation was 32% (32 out of 100) and 29% (85 out of 295) among INH-susceptible isolates and INH-resistant or -intermediate isolates, respectively. Sequencing of five INH-susceptible isolates with such mutations showed that all five had the Arg463Leu mutation. We conclude that the Arg463Leu mutation of katG of M. tuberculosis is not a reliable indicator of INH resistance.     

New real-time PCR able to detect in a single tube multiple rifampin resistance mutations and high-level isoniazid resistance mutations in Mycobacterium tuberculosis

The emergence of resistance to antituberculosis drugs is a relevant matter worldwide, but the retrieval of antibiograms for Mycobacterium tuberculosis is severely delayed when phenotypic methods are used. Genotypic methods allow earlier detection of resistance, although conventional approaches are cumbersome or lack sensitivity or specificity. We aimed to design a new real-time PCR method to detect rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains in a single reaction tube. First, we characterized the resistant isolates in our area of Spain by DNA sequencing. Some mutation was found within the rpoB core region in all the RIF-resistant (RIF(r)) strains. Forty-six percent of the INH-resistant (INH(r)) strains showed a mutation in katG codon 315, and most of these were associated with high MICs. Eighteen of the RIF(r), INH(r), and multidrug-resistant strains sequenced were tested by our real-time PCR assay; and full concordance of the results of the PCR with the sequencing data was obtained. In addition, a blind test was performed with a panel of 15 different susceptible and resistant strains from throughout Spain, and our results were also in 100% agreement with the sequencing data. Ours is the first assay based on rapid-cycle PCR able to simultaneously detect in a single reaction tube a large variety of mutations associated with RIF resistance (12 different mutations affecting 8 independent codons, including the most prevalent mutations at positions 526 and 531) and the most frequent INH resistance mutations. Our design could be a model for new, rapid genotypic methods able to simultaneously detect a wide variety of antibiotic resistance mutations.     

Mutation detection and accurate diagnosis of extensively drug-resistant tuberculosis: report from a tertiary care center in India

We screened and spoligotyped 150 consecutive phenotypically confirmed extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) isolates (January 2008 to March 2009) for rifampin, isoniazid, fluoroquinolone, and aminoglycoside resistance targeting rpoB, inhA, katG, gyrA, gyrB, and rrs. Mutations predominant among XDR-TB were S315T (katG) (100% of isolates), S531L (rpoB) (97% of isolates), D94G (gyrA) (53% of isolates), and A1401G (rrs) (71% of isolates). Spoligotyping revealed 62% of the isolates to be Beijing.     

Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens

The new GenoType MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany) was tested with 125 clinical isolates and directly with 72 smear-positive sputum specimens for its ability to detect rifampin (RMP) and isoniazid (INH) resistance in Mycobacterium tuberculosis complex (MTBC) strains. In total, 106 RMP(r)/INH(r), 10 RMP(s)/INH(r), and 80 RMP(s)/INH(s) MTBC strains were comparatively analyzed with the new and the old MTBDR assays. Besides the detection of mutations within the 81-bp hot spot region of rpoB and katG codon 315, the GenoType MTBDRplus assay is designed to detect mutations in the regulatory region of inhA. The applicability of the new assay directly to specimens was shown, since 71 of 72 results for smear-positive sputa and all 125 results for clinical isolates were interpretable and no discrepancies compared with the results of real-time PCR or DNA sequencing were obtained. In comparison to conventional drug susceptibility testing, both assays were able to identify RMP resistance correctly in 74 of 75 strains (98.7%) and 30 of 31 specimens (96.8%). The misidentification of RMP resistance was obtained for two strains containing rpoB P533L mutations. Compared to the old MTBDR assay, the new GenoType MTBDRplus assay enhanced the rate of detection of INH resistance from 66 (88.0%) to 69 (92.0%) among the 75 INH-resistant strains and 36 (87.8%) to 37 (90.2%) among the 41 specimens containing INH-resistant strains. Thus, the new GenoType MTBDRplus assay represents a reliable and upgraded tool for the detection of INH and RMP resistance in strains or directly from smear-positive specimens.     

Biological and molecular characteristics of Mycobacterium tuberculosis clinical isolates with low-level resistance to isoniazid in Japan

We reevaluated the BACTEC MGIT 960 antimicrobial susceptibility testing system (MGIT 960 AST) by using 1,112 isolates of Mycobacterium tuberculosis. When the results of MGIT 960 AST were compared with that of the proportion method using Ogawa medium (Ogawa PM), discrepant results were obtained for 30 strains with isoniazid, all resistant by MGIT 960 AST but susceptible by Ogawa PM. For 93% of the strains that produced discrepant results, the MIC was 0.4 or 0.8 microg/ml, showing resistance by the proportion method using Middlebrook agar plates. Furthermore, it was also established by analyses of the katG and inhA genes that strains resistant only by MGIT 960 AST have a low level of isoniazid (INH) resistance, indicating that MGIT 960 AST is a reliable method. Ninety-six strains were resistant to 0.1 microg/ml INH by MGIT 960 AST. When they were divided into three groups, Low-S (susceptible at 0.2 microg/ml), Low-R (resistant at 0.2 microg/ml), and High-R (resistant at 1.0 microg/ml), by Ogawa PM, 43.3% of the Low-S strains had mutations in the promoter region of inhA and no mutations were detected in katG codon 315, while 61.7% of the High-R strains had katG codon 315 mutations or a gross deletion of katG. These results suggest that mutations in inhA are associated with low-level resistance to INH and katG codon 315 mutations are associated with high-level resistance to INH. In addition, the analyses demonstrated some relationship of mutations in the inhA gene with ethionamide resistance for the Low-S strains, but not for the High-R strains.     

Magnitude of Gene Mutations Conferring Drug Resistance in Mycobacterium Tuberculosis Isolates from Lymph Node Aspirates in Ethiopia

Objective: Resistance to drugs is due to particular genomic mutations in the specific genes of Mycobacterium tuberculosis. Timely genetic characterization will allow identification of resistance mutations that will optimize an effective antibiotic treatment regimen. We determine the magnitude of gene mutations conferring resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among tuberculosis (TB) lymphadenitis patients.Methods: A cross sectional prospective study was conducted among 226 M.tuberculosis isolates from culture positive lymph node aspirates collected from TB lymphadenitis patients between April 2012 and May 2012. Detection of mutations conferring resistance to drugs was carried out using GenoType^® MTBDRplus and GenoType® MTBDRsl assay.Results: Out of the 226 strains, mutations conferring resistance to INH, RMP, multidrug resistance tuberculosis (MDR-TB) and EMB were 8, 3, 2 and 2 isolates, respectively. There was no isolated strain that showed mutation in the inhA promoter region gene. All INH resistant strains had mutations in the katG gene at codon 315 with amino acid change of S315T1. Among rifampicin resistant strains, two isolates displayed mutations at codon 531 in the rpoB gene with amino acid change of S531L and one isolate was by omission of wild type probes at Q513L. According to mutations associated with ethambutol resistance, all of the isolates had mutations in the embB gene with aminoacid change of M306I. All isolates resistant to INH, RMP and MDR using BacT/AlerT 3D system were correctly identified by GenoType® MTBDRplus assay.Conclusion: We observed mutations conferring resistance to INH at S315T1 of the katG gene, RMP at S531L and Q513L in the rpoB genes and EMB at M306I of the embB gene. In the absence of conventional drug susceptibility testing, the effort to develop easy, rapid and cost effective molecular assays for drug resistance TB monitoring is definitely desirable and the GenoType® MTBDRplus assay was found to be a useful method for diagnosis of resistance to INH, RMP and MDR from lymph node aspirates. Further molecular cluster analysis to determine transmission dynamics of mutated strain is required.     

Detection of isoniazid-resistant Mycobacterium tuberculosis strains by a multiplex allele-specific PCR assay targeting katG codon 315 variation

We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the katG gene codon 315, including AGC-->ACC and ACA (Ser-->Thr) substitutions that confer resistance to isoniazid (INH) in Mycobacterium tuberculosis clinical isolates. The 315 ACC allele is found in the majority of Inh(r) strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States. The mutations in katG315 are revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. Initially optimized on the purified DNA samples, the assay was then tested on crude cell lysates and auramine-stained sputum slide preparations with the same reproducibility and interpretability of profiles generated by agarose gel electrophoresis. The MAS-PCR assay can be used for the detection of resistance to INH in clinical laboratories in regions with a high prevalence of MDR M. tuberculosis strains.     

Molecular epidemiology and prevalence of mutations conferring rifampicin and isoniazid resistance in Mycobacterium tuberculosis strains from the southern Ukraine

Understanding the molecular epidemiology of tuberculosis (TB) and mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. A structured questionnaire was used to collect basic epidemiological data from 589 patients with radiologically confirmed TB in the Odessa and Nikolaev regions of the Ukraine in 2003-2004. A non-commercial reverse hybridisation assay and DNA sequencing were used to detect mutations associated with rifampicin and isoniazid resistance. Genotyping was performed using multilocus variable number tandem repeat (VNTR) typing and spoligotyping. Mutations conferring rifampicin and isoniazid resistance were detected in 32.9% and 44.0%, respectively, of 225 Mycobacterium tuberculosis isolates from individual consecutive patients. Mutations in codon 531 and codon 315 of the rpoB and katG genes, respectively, were predominant among drug-resistant isolates. Multidrug (MDR) resistance rates were significantly higher among former prison inmates compared with non-prisoners (54.8% vs. 27.3%; RR 2.01; 95% CI 1.35-2.97) and the prevalence of mutations was higher in Beijing strains sharing the VNTR signature 223325173533424 than in other Beijing strains (71.4% vs. 45.7%; RR 1.74; 95% CI 1.17-2.57), suggesting that this group may be responsible for rapid transmission of MDR TB in the southern Ukraine.     

Clinical and genotypic characteristics of childhood tuberculosis in Chongqing, China

The aims of this study were to assess the prevalence of isolates of Beijing lineage in childhood tuberculosis (TB) and the association between strain family and clinical characteristics. A total of 105 children aged less than 18 years with culture-confirmed TB were evaluated by spoligotyping and the 12-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) scheme. Of 105 isolates spoligotyped, the major spoligotypes found were Beijing plus Beijing-like lineages (64.8%), followed by T1 lineages (12.3%) and MANU2 lineages (5.7%). MIRU typing identified 72 types that defined 17 clusters and 55 unique isolates. The largest cluster was 223325173533, which belongs to the Beijing lineage. Children in rural areas accounted for 68.8% and 50.5% of the children with TB were older than 5 years. Extrapulmonary tuberculosis accounted for 67.6%, and tuberculous meningitis (TBM) 53.3%. There was no association with clinical characterization (P>0.05). In conclusion, Beijing isolates are the dominant genotype family in children in China. Beijing and non-Beijing strains did not differ in their propensities to cause extrapulmonary tuberculosis in general or meningitis in particular and there was no significant difference between children infected with Beijing strains and those infected with non-Beijing strains with respect to the clinical features.