Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolates from China
31
Citation
59
Reference
10
Related Paper
Citation Trend
Abstract:
ABSTRACT To investigate the molecular characterization of multidrug-resistant tuberculosis (MDR-TB) isolates from China and the association of specific mutations conferring drug resistance with strains of different genotypes, we performed spoligotyping and sequenced nine loci ( katG , inhA , the oxyR-ahpC intergenic region, rpoB , tlyA , eis , rrs , gyrA , and gyrB ) for 128 MDR-TB isolates. Our results showed that 108 isolates (84.4%) were Beijing family strains, 64 (59.3%) of which were identified as modern Beijing strains. Compared with the phenotypic data, the sensitivity and specificity of DNA sequencing were 89.1% and 100.0%, respectively, for isoniazid (INH) resistance, 93.8% and 100.0% for rifampin (RIF) resistance, 60.0% and 99.4% for capreomycin (CAP) resistance, 84.6% and 99.4% for kanamycin (KAN) resistance, and 90.0% and 100.0% for ofloxacin (OFX) resistance. The most prevalent mutations among the MDR-TB isolates were katG315 , inhA15 , rpoB531 , - 526 , and - 516 , rrs1401 , eis-10 , and gyrA94 , - 90 , and - 91 . Furthermore, there was no association between specific resistance-conferring mutations and the strain genotype. These findings will be helpful for the establishment of rapid molecular diagnostic methods to be implemented in China.Keywords:
INHA
rpoB
Capreomycin
Kanamycin
Intergenic region
Two hundred and seventy-eight M. tuberculosis DNA samples taken from patients with clinically confirmed pulmonary and extrapulmonary tuberculosis were studied. Mutations of the rpoB, inhA, katG, and ahpC genes were analyzed by using multiple drug-resistant (MDR) biochips. A hundred and twenty-nine (46%) rifampicin- and isoniazid-sensitive strains and 149 (54%) resistant ones were detected. Out of the 149 drug-resistant strains, resistance to one drug (rifampicin or isoniazid) was revealed in 7 (4.7%) and 48 (32.3%) cases, respectively. The strains simultaneously resistant to both drugs were detected in 94 (63%) cases. In the Republic of Kyrghyzstan, patients with drug-resistant pulmonary tuberculosis were observed to have more commonly multidrug-resistant strains (63%) than the strains resistant to one drug (rifampicin or isoniazid). In this republic, the main cause of rifampicin resistance of Mycobacterium tuberculosis is the Ser531-Leu mutation of the rpoB gene in codon 531 and the Ser315-->Thr of the katG gene in codon 315.
rpoB
INHA
Cite
Citations (4)
INHA
rpoB
Cite
Citations (25)
Resistance to antimycobacterial agents consistently remains a major obstacle to end TB in India. Geographical prevalence data regarding drug-resistant evolutionary genetics of M. tuberculosis (MTB) remains sparse in India. Our objective was to determine the genotypic drug resistance mutation pattern for Rifampicin and Isoniazid of MTB isolates to gain an understanding of the prevailing molecular epidemiology of drug-resistant tuberculosis. In this study 2528 M. tuberculosis DNA isolates from presumptive DRTB suspects received at the nodal TB reference laboratory in Kerala were tested for Rifampicin and Isoniazid resistance by sequence-based diagnostic Line Probe assay (LPA). Geographical prevalence and associations of rpoB, katG, inhA resistance codons was analyzed from January 2019 to March 2020. Among the 2528 DNA samples subjected for Rifampicin and Isoniazid resistance determination by LPA, 146 (5.8%) isolates were resistant to both drugs. Isoniazid mono-resistance was found in 164 (6.5%) and Rifampicin mono-resistance in 38 (1.5%) isolates. The most frequent rpoB mutation was S531L (60.32%) followed by S531W/L533P mutations seen in 8.15% of the isolates. S315T1 KatG mutation was seen in 97.33% of Isoniazid resistant isolates. 84.68% isolates with rpoB S531L mutation were found to be multidrug-resistant. 82.9% of isolates with rpoB S531L mutation showed katG S315T1 mutation. Mono isoniazid-resistant isolates were significantly higher compared to mono rifampicin-resistant isolates among the DNA isolates studied in our region. The molecular epidemiological pattern most frequently associated with multidrug resistance was rpoB S531L which was significantly associated with the co-presence of S315T1 mutation.
rpoB
INHA
Ethionamide
Cite
Citations (3)
Objective: The objective of our study was to evaluate the use of a real-time polymerase chain reaction (PCR)-based technique for the prediction of phenotypic resistance of Mycobacterium tuberculosis. Materials and Methods: We tested 67 M tuberculosis strains (26 drug resistant and 41 drug susceptible) using a method recommended for the LightCycler platform. The susceptibility testing was performed by the absolute concentration method. For rifampin resistance, two regions of the rpoB gene were targeted, while for identification of isoniazid resistance, we searched for mutations in katG and inhA genes. Results: The sensitivity and specificity of this method for rapid detection of mutations for isoniazid resistance were 96% (95% CI: 88% to 100%) and 95% (95% CI: 89% to 100%), respectively. For detection of rifampin resistance, the sensitivity and specificity were 92% (95% CI: 81% to 100%) and 74% (95% CI: 61% to 87%), respectively. The main isoniazid resistance mechanism identified in our isolates is related to changes in the katG gene that encodes catalase. We found that for rifampin resistance the concordance between the predicted and observed phenotype was less than satisfactory. Conclusions: Using this method, the best accuracy for genotyping compared with phenotypic resistance testing was obtained for detecting isoniazid resistance mutations. Although real-time PCR assay may be a valuable diagnostic tool, it is not yet completely satisfactory for detection of drug resistance mutations in M tuberculosis.
rpoB
INHA
Concordance
Cite
Citations (18)
Tuberculosis is still a global emergency and a major public health problem, in some cases related to the appearance of strains of multi drug resistance (MDR) and extensive drug resistance (XDR) Mycobacterium tuberculosis complex.The correct determination of antibiotic sensitivity profiles is therefore crucial to carry out appropriate treatment aimed to decrease the infectivity of each patient and to reduce mortality. The poor adherence to treatment by the patient or the use of therapies based on a single drug, as a result of incorrect requirements, promote the development of drug-resistance. Have some time on the market of molecular diagnostic tests that allow, quickly and directly from biological sample to search for resistance genes some key drugs of anti-TB therapy (Rifampicin and Isoniazid). One of the tests in question is the Genotype MTBDRplus ver 2.0 which can reveal the presence of genes for resistance to Isoniazid (INH) and Rifampin (RMP).The loci analyzed are those corresponding to the rpoB gene for rifampicin, katG and inhA for isoniazid. Our study is based on the analysis of 83 strains of tubercular Mycobacteria identified and isolated from patients with tuberculosis disease and subjected to the tests sensitivity, searching for mutations and phenotypic susceptibility testing for Rifampicin and Isoniazid.The comparison of the results has shown that the results obtained using the Genotype MTBDRplus ver 2.0 test, were similar to the results obtained by the traditional susceptibility testing.
INHA
rpoB
Cite
Citations (0)
We have developed a high-resolution melting (HRM) assay to scan for mutations in the rpoB, inhA, ahpC, and katG genes and/or promoter regions for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis. For assay development, 23 drug-resistant isolates of M. tuberculosis having 29 different mutations, together with 40 drug-susceptible isolates, were utilized. All 29 mutations were accurately detected by our assay. We further validated the assay with a series of 59 samples tested in a blind manner. All sequence alterations that were within the regions targeted by the HRM assay were correctly identified. Compared against results of DNA sequencing, the sensitivity and specificity of our HRM assay were 100%. For the blinded samples, the specificities and sensitivities were 89.3% and 100%, respectively, for detecting rifampin resistance and 98.1% and 83.3%, respectively, for detecting isoniazid resistance, as isolates with mutations in regions not encompassed by our assay were not detected. A C-to-T sequence alteration at position -15 of the ahpC regulatory region, which was previously reported to be associated with isoniazid resistance, may possibly be a polymorphism, as it was detected in an isoniazid-susceptible M. tuberculosis isolate. HRM is a rapid, accurate, simple, closed-tube, and low-cost method. It is thus an ideal assay to be used in countries with a high prevalence of drug-resistant M. tuberculosis and where cost-effectiveness is essential. As a mutation-scanning assay for detecting drug-resistant M. tuberculosis, it can potentially lead to better treatment outcomes resulting from earlier treatment with the appropriate antibiotics.
High Resolution Melt
Cite
Citations (49)
Rapid and accurate identification of Mycobacterium tuberculosis and its fast detection of rifampicin and isoniazid's drug-resistance gene mutations had an important guiding significance on the diagnosis and treatment of tuberculosis patients.In this paper,according to the sequence of H37RV strain,sixteen oligonucleotide probes were designed specifically to detect the mutant sequences in rpoB,katG and inhA gene to identify the result of the drug resistance,and at the same time,methodology evaluation was performed.
rpoB
INHA
Cite
Citations (0)
Ontario bears the greatest burden of tuberculosis in Canada, with 40% of all cases and 60% of multidrug-resistant cases. The purpose of this study was to genotypically characterize isoniazid- and rifampicin-resistant isolates and compare these results with phenotypic drug susceptibility testing data. This is the first Canadian study to examine gene mutations that contribute to multidrug-resistant tuberculosis. A total of 751 tuberculosis isolates were tested for drug resistance using phenotypic antimicrobial susceptibility testing methods. Isolates were then characterized using molecular methods. Following DNA extraction, PCR amplification and sequence analysis were performed on the rifampicin resistance region of rpoB, as well as the region surrounding katG315 and the inhA promoter region associated with isoniazid resistance. Eighteen different mutation types were found in the rpoB region of rifampicin-resistant isolates. Isolates with mutations at residues rpoB531 (64.1%), rpoB526 (15.2%) and rpoB516 (8.7%) were the most common. In addition, an insertion was found at residue 514. Three phenotypically rifampicin-resistant isolates (3.3%) were genotypically wild-type. In isoniazid-resistant strains, mutations were found most commonly at katG315 (45.4%) as well as at the inhA promoter region (28.6%). Thirty-nine isolates (25.3%) were phenotypically isoniazid-resistant but genotypically wild-type. The katG315 mutation was statistically associated with multidrug-resistant isolates. This study expands the knowledge of mutations that potentially contribute to drug resistance in tuberculosis and lays the foundation for developing molecular-based tests to determine drug resistance in clinical tuberculosis isolates.
rpoB
INHA
Cite
Citations (36)
ABSTRACT Vietnam is ranked 13th among the WHO list of 22 high-burden countries, based upon estimated total number of tuberculosis cases. Despite having a model national tuberculosis program, consistently achieving and exceeding WHO targets for detection and cure, drug-resistant and multidrug-resistant tuberculosis cases continue to rise. Rapid multidrug-resistant tests applicable in this setting, coupled with effective treatment regimens, would be a useful tool in reversing this trend, allowing early identification of patients with multidrug-resistant tuberculosis and avoiding resistance-amplifying regimens. Sequencing of consecutive isolates identified by the National Tuberculosis Program showed 89% of isoniazid-resistant isolates could be detected by targeting just 2 codons, katG 315 and −15C→T in the inhA promoter, while rifampin resistance will be more complex to detect, with many different mutation and insertion events in rpoB . The most prevalent rifampin resistance-conferring mutations, as in other countries, were in rpoB codons 531 (43%), 526 (31%), and 516 (15%). However, a hybridization-based resistance test with probes targeting the 5 most common mutations would only detect 78% of rifampin-resistant isolates. Overall, these data suggest that rifampin resistance may be used as a surrogate marker for multidrug-resistant tuberculosis and that a sensitivity of between 70 to 80% may be possible for rapid molecular detection of multidrug-resistant tuberculosis in this setting.
rpoB
INHA
Cite
Citations (98)
Role of line probe assay in diagnosis and detection of drug resistance in Mycobacterium tuberculosis
Tuberculosis caused by Mycobacterium tuberculosis has remained a major global health problem worldwide. TB requires prolonged period of time for isolation by conventional culture methods. The emergence and spread of multi drug resistant (MDR-TB) poses great threats and challenges in controlling the infection. MDR-TB is resistant to both first line drugs rifampicin and isoniazid. PCR tests are based on targeting the mutation in rpoB, katG and inhA genes which can detect resistance to these drugs. To compare microscopy, conventional culture and Line probe assay for the detection of M. tuberculosis & detect rifampicin and isoniazid resistance using Lineprobe assay in various clinical samples. A total of 347 suspected patients of tuberculosis were included in the study. Demographic details & clinical presentation was noted. Various samples were received & processed for ZN staining, culture on LJ media and Line probe assay. Out of 347 cases, majority of cases were in the age group of 51-60 years (18.4%). Majority of the population was males (65.1%). Among suspected tuberculosis patients, cough with expectoration (55.9%) was the commonest complaint. Microscopy was positive in 17.3%, conventional culture was positive in 16.1% and line probe assay was positive in 26.2%. Out of 347, 91 were diagnosed with MTB, out of which 85.7% were sensitive to both rifampicin and isoniazid whereas 14.3% showed resistance to either rifampicin / isoniazid or both. LPA & direct microscopy are a good screening method for early diagnosis and detection of drug resistance but are not a complete replacement of conventional culture which is still a gold standard.
Cite
Citations (1)