Mutations in the rpoB and katG genes leading to drug resistance in Mycobacterium tuberculosis in Latvia

To characterize the genetic basis of drug resistance in Mycobacterium tuberculosis in Latvia, mutations involved in rifampin (rpoB gene) and isoniazid (katG gene) resistance in DNA from 19 drug-susceptible and 51 multidrug-resistant M. tuberculosis complex isolates were analyzed. The most frequent rpoB gene mutations found by the Line Probe assay were the S531L (14 of 34 isolates), D516V (7 of 34), H526D (4 of 34), and D516Y plus P535S (4 of 34) mutations. Direct sequencing of seven isolates with unclear results from Line Probe assay showed the presence of the L533P mutation and the Q510H plus H526Y (1 of 34) and D516V plus P535S (4 of 34) double mutations, neither of which has been described previously. Single-strand conformation polymorphism analysis showed strand mobility differences between the rifampin-susceptible and -resistant samples for the D516V, H526D, and D516Y plus P535S mutations but not for the S531L mutation. Nucleotide substitution at codon 315 (AGC-->ACC) of the katG gene was found in 48 of 51 multidrug-resistant samples by sequencing. Furthermore, katG gene restriction fragment length polymorphism analysis with endonuclease AciI confirmed the nucleotide change in codon 315.     

rpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy

Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser-->Leu) and 11 (29.7%) had GAC in position 526 (His-->Asp).     

结核分枝杆菌耐利福平rpoB基因突变的序列分析

目的探讨结核分枝杆菌耐药基因rpoB突变与利福平耐药性的关系。方法采集81例临床标本,分离结核分枝杆菌菌株,PCR扩增rpoB基因及测序,并与利福平药物敏感试验结果比较。结果27例耐利福平菌株中,20株发生rpoB基因突变(74.1%),突变位点包括531和526在内的7个位点10种突变类型,并发现新的突变位点和突变形式;54株敏感株中,1株发生突变(1.9%)。结论利福平耐药rpoB基因突变有一定区域性,为发展快速的基因诊断技术检测耐多药结核病奠定了基础。     

Rapid detection of rpoB gene mutations conferring rifampin resistance in Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute.     

Evaluation of the rpoB macroarray assay to detect rifampin resistance in Mycobacterium tuberculosis in Beijing, China

The aim of this study was to evaluate the modified rifampin oligonucleotide (RIFO) macroarray method to detect mutations in the hot-spot region of the rpoB gene, associated with rifampin (RIF) resistance in Mycobacterium tuberculosis. The study sample included 123 strains of M. tuberculosis isolated in the Beijing, China, area in 2002–2005, including 73 RIF-resistant and 40 pansusceptible strains. The genotypic assay successfully identified 91.8% of the RIF-resistant strains, whereas no mutations were found in RIF-susceptible strains. The most frequently detected rpoB mutations were in the codons 516, 526, and 531, together accounting for 74% of RIF-resistant strains. Spoligotyping subdivided all strains into 11 unique profiles and 3 profiles shared by 3, 4, and 103 strains, respectively. The 113 strains belonged to the Beijing family genotypes, defined by the specific spoligotype signature (absence of signals 1–34) and deletion of the RD105 region. The rpoB S531L (TCG→TTG) mutation was found in 57.4% of the RIF-resistant strains of the Beijing genotype. A mutation in the rpoB hot-spot region was found in 51 of the 55 (92.7%) multidrug-resistant strains (i.e., resistant to at least RIF and isoniazid), thus demonstrating the added utility of the modified RIFO method to predict multidrug resistance. The RIFO method is relatively simple to perform and allows straightforward interpretation of results; consequently, it can be used in clinical diagnostic laboratories as a fast complement to phenotypic methods.     

Distribution of rpoB mutations among multidrug-resistant Mycobacterium tuberculosis (MDRTB) strains from Thailand and development of a rapid method for mutation detection.

Since rifampicin resistance is a surrogate marker for multidrug-resistant Mycobacterium tuberculosis (MDRTB), the present study aimed to investigate rpoB mutations conferring rifampicin resistance in M. tuberculosis strains from Thailand, and to develop a rapid, inexpensive and simple PCR-based method for rapid detection of MDRTB. Overall, 267 M. tuberculosis isolates, including 143 MDRTB isolates, were investigated. Isolates of the Beijing strain predominated among the MDRTB isolates (79.1%), but accounted for only 45.5% of the susceptible isolates. Mutations in the rpoB gene were found most commonly at codons 531, 526 and 516 (58%, 25.2% and 9.1%, respectively). A multiplex allele-specific PCR was developed and tested with 216 clinical isolates. In comparison with the proportion method, the method showed 94.2% sensitivity and 100% specificity, and had a 100% positive predictive value and a 95% negative predictive value, which suggested that this method could be useful for screening for MDRTB, particularly in resource-limited countries.     

Mutations in rpoB gene and rifabutin susceptibility of multidrug-resistant Mycobacterium tuberculosis strains isolated in Australia.

Control of tuberculosis, the single largest killer among the infectious diseases, has been threatened by the emergence of multidrug-resistant Mycobacterium tuberculosis (MDRTB) infection due to the limited treatment options. Rifampicin (RIF) resistance is considered as a marker for MDRTB. The aim of this study was the detection of rpoB gene mutations and rifabutin resistance in MDRTB strains recently isolated in Australia by a line probe assay (INNO-LiPA Rif. TB, Innogenetics). Rifabutin and RIF susceptibility of 20 MDRTB and 16 RIF-sensitive M. tuberculosis complex clinical isolates were studied. The overall concordance of the line probe assay (LiPA) with phenotypic RIF susceptibility test was 96%. Seven distinct nucleotide substitutions were identified in 21 of 22 RIF-resistant isolates of diverse geographical origins, but in none of the RIF-sensitive strains. The majority (71%) of mutations occurred in the 526-533 codons and were associated with resistance to rifabutin and RIF. Of the RIF-resistant MDRTB strains, 18% appeared to be rifabutin-sensitive and produced delta S2 and delta S3 INNO-LiPA patterns. We conclude that amino acid substitutions at Asp516 and Ser522 in the rpoB gene in RIF-resistant M. tuberculosis predict rifabutin susceptibility for MDRTB. Use of the LiPA for RIF and rifabutin resistance may facilitate the rapid response required to limit the extent and severity of MDRTB transmission and infection.     

Detection of rpoB mutations in Mycobacterium tuberculosis by biprobe analysis

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

Multidrug resistance to Mycobacterium tuberculosis in a tertiary hospital

OBJECTIVE: The magnitude of drug-resistant Mycobacterium tuberculosis infection (MDR-TB) in Nigeria, the most populous country in sub-Saharan Africa, is largely unknown. This information would assist policymakers to develop intervention strategies against tuberculosis (TB) in the country. MATERIALS AND METHODS: This is a one-year laboratory-based study. Specimens from suspected new TB patients sent to the TB laboratory of the Department of Medical Microbiology, University College Hospital Ibadan, Nigeria from May 1, 2005 to April 27, 2006 were processed and analyzed. The specimens were stained with Ziehl-Neelsen (Z-N) reagents and cultured on Lowenstein-Jensen medium, incubated at 37 degrees C for 6-8 weeks. Isolates were confirmed as MDR-TB by Z-N reactions and biochemical methods. Drug susceptibility to streptomycin, ethambutol, rifampicin and isoniazid was done using Bactec 460 TB radiometric method. RESULTS: Of the 1,120 specimens processed, 80 (7.1%) were smear positive, while 56 (5.0%) were culture positive, even though the association was not statistically significant (p > 0.05). Culture contamination rate was 8.8%. Thirty (53.6%) of the culture positive isolates were resistant to both isoniazid and rifampicin, while 26 (46.4%) were susceptible. About half--53.3%--of the resistant isolates were from the antiretroviral clinic, while 10 (33.4%) were from peripheral centers. CONCLUSION: This study shows that MDR-TB is emerging in Nigeria. Further studies on MDR-TB are urgently needed in the country to ascertain the magnitude of the problem and to proffer solutions to it.     

Drug resistance and genotypic analysis of Mycobacterium tuberculosis strains from Thai tuberculosis patients

The aim of this study was the molecular characterization of primary drug‐resistant Mycobacterium tuberculosis strains in Thailand. We examined a group of M. tuberculosis isolates from newly registered tuberculosis (TB) cases, collected at the largest university hospital, the Siriraj Hospital, in Thailand. Of 76 selected drug‐resistant M. tuberculosis strains recovered from previously untreated pulmonary TB patients whose sputum samples were sent to this hospital, 29 (38%) were single‐drug resistant, 26 (34%) multidrug resistant and two (2.6%) extensively drug resistant. Fifty (66%) strains belonged to Beijing genotype. The study demonstrate a severe problem of drug resistance among recently detected TB patients, and two large clusters of genetically similar strains indicated ongoing transmission of drug‐resistant TB.     

In-vitro activity of rifabutin against rifampicin-resistant Mycobacterium tuberculosis isolates with known rpoB mutations

The relationship between resistance to rifampicin and rifabutin and genetic alterations in the rpoB gene of 41 rifampicin-resistant isolates of Mycobacterium tuberculosis was evaluated. Although 35 isolates with rifampicin MICs > or = 32 mg/L were also rifabutin-resistant, six isolates with rifampicin MICs of 2-16 mg/L were susceptible to rifabutin (MIC < or = 0.5 mg/L). Mutations Asp516Val, Asp516Tyr, Leu533Pro and the double mutation Met515Ile and Leu533Pro influenced susceptibility to rifampicin, but not to rifabutin. All mutations at codons 531 and 526, except one isolate with a His526Cys mutation, correlated with resistance to both compounds.     

Mixed infection with Beijing and non-Beijing strains and drug resistance pattern of Mycobacterium tuberculosis

Mixed infection with Beijing and non-Beijing strains of Mycobacterium tuberculosis has been reported and has been suggested to mediate elevation of the reinfection rate in regions with a high incidence of tuberculosis (TB). To evaluate the prevalence of infection with both Beijing and non-Beijing strains of M. tuberculosis in eastern Taiwan, the region with the highest TB incidence in Taiwan, 185 active pulmonary TB patients were enrolled at Tzu Chi General Hospital from October 2007 to September 2008. A modified multiplex PCR method was developed to distinguish Beijing and non-Beijing strains directly using the sputum of patients. Of the 185 patients, 46.5% were infected with a Beijing strain, 42.2% were infected with a non-Beijing strain, and 11.3% were infected with both strain types. Notably, mixed infection with both strain types was not associated with TB treatment history or the high-incidence race group, aborigines. In addition, the incidence rate of mixed infection before treatment with anti-TB medication was as high as that in patients with a history of anti-TB treatment. Further analysis of antibiotic susceptibility revealed that Beijing strains alone had the highest multidrug resistance rate (17.5%), mixed infection had the highest rate of resistance to at least one drug (23.8%), and non-Beijing strains had the highest rate of sensitivity to all drugs (79.5%), implying that Beijing strains are predominant in the development of drug resistance in tuberculosis.     

Newly developed primers for comprehensive amplification of the rpoB gene and detection of rifampin resistance in Mycobacterium tuberculosis

New rpoB gene primers for detecting Rif(r) in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample. Of the 99 Rif(r) isolates examined, 97% had mutations within cluster I, 2% at codon 176, and 1% at codon 497.     

Rapid detection of rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates in shanghai by using the amplification refractory mutation system

Rapid detection of drug resistance in Mycobacterium tuberculosis is essential for efficient treatment and control of this pathogen. The amplification refractory mutation system (ARMS) was used to detect mutations in the rifampin resistance-determining region of the rpoB gene. A total of 39 rifampin-resistant M. tuberculosis isolates in Shanghai were analyzed by this assay, resulting in 92.3% sensitivity (36 of 39) and 87.2% concordance (34 of 39) relative to DNA sequencing, by which 41 mutations of 11 different types, including 9 point mutations and 2 deletions, were identified in the rpoB gene. The most frequent mutations were those associated with codon 531 (21 of 39 [53.8%]) and codon 526 (9 of 39 [23.1%]). The results suggest that the ARMS assay is rapid and simple to implement and could be performed for detection of rifampin resistance in M. tuberculosis to complement conventional culture-based methods.     

Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China

Mutations in the 81-bp rifampin resistance determining region (RRDR) and mutation V176F locating at the beginning of the ropB gene were analyzed by DNA sequencing of 86 Mycobacterium tuberculosis clinical isolates (72 resistant and 14 sensitive) from different parts of China. Sixty-five mutations of 22 distinct kinds, 21 point mutations, and 1 insertion were found in 65 of 72 resistant isolates. The most common mutations were in codons 531 (41%), 526 (40%), and 516 (4%). Mutations were not found in seven (10%) of the resistant isolates. Six new alleles within the RRDR, along with five novel mutations outside the RRDR, are reported. None of isolates contained the V176 mutation.     

Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from Brazil

Mutations in a 69-bp region of the rpoB gene associated with rifampin resistance (Rif(r)) in 100 isolates (82 Rif(r)) from three states of Brazil were studied. Twenty-one different kinds of mutations were identified in the Rif(r) isolates, and six new alleles are described.     

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

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