inhA Promoter Region Research Articles

Diagnostic performance of the GenoType MTBDRplus VER 2.0 line probe assay for the detection of isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

Isoniazid (INH) resistant Mycobacterium tuberculosis (Hr-TB) is the most common type of drug resistant TB, and is defined as M tuberculosis complex (MTBC) strains resistant to INH but susceptible to rifampicin (RIF). Resistance to INH precedes RIF resistance in almost all multidrug resistant TB (MDR-TB) cases, across all MTBC lineages and in all settings. Therefore, early detection of Hr-TB is critical to ensure rapid initiation of appropriate treatment, and to prevent progression to MDR-TB. We assessed the performance of the GenoType MTBDRplus VER 2.0 line probe assay (LPA) in detecting isoniazid resistance among MTBC clinical isolates. A retrospective study was conducted among M. tuberculosis complex (MTBC) clinical isolates obtained from the third-round Ethiopian national drug resistance survey (DRS) conducted between August 2017 and December 2019. The sensitivity, specificity, positive predictive value, and negative predictive value of the GenoType MTBDRplus VER 2.0 LPA in detecting INH resistance were assessed and compared to phenotypic drug susceptibility testing (DST) using the Mycobacteria Growth Indicator Tube (MGIT) system. Fisher's exact test was performed to compare the performance of LPA between Hr-TB and MDR-TB isolates. A total of 137 MTBC isolates were included, of those 62 were Hr-TB, 35 were MDR-TB and 40 were INH susceptible. The sensitivity of the GenoType MTBDRplus VER 2.0 for detecting INH resistance was 77.4% (95% CI: 65.5-86.2) among Hr-TB isolates and 94.3% (95% CI: 80.4-99.4) among MDR-TB isolates (P = 0.04). The specificity of the GenoType MTBDRplus VER 2.0 for detecting INH resistance was 100% (95% CI: 89.6-100). The katG 315 mutation was observed in 71% (n = 44) of Hr-TB phenotypes and 94.3% (n = 33) of MDR-TB phenotypes. Mutation at position-15 of the inhA promoter region alone was detected in four (6.5%) Hr-TB isolates, and concomitantly with katG 315 mutation in one (2.9%) MDR-TB isolate. GenoType MTBDRplus VER 2.0 LPA demonstrated improved performance in detecting INH resistance among MDR-TB cases compared to Hr-TB cases. The katG315 mutation is the most common INH resistance conferring gene among Hr-TB and MDR-TB isolates. Additional INH resistance conferring mutations should be evaluated to improve the sensitivity of the GenoType MTBDRplus VER 2.0 for the detection of INH resistance among Hr-TB cases.

Frequency of rpoB, katG, and inhA Gene Polymorphisms Associated with Multidrug-Resistant Mycobacterium tuberculosis Complex Isolates among Ethiopian TB Patients: A Systematic Review.

Tuberculosis (TB) is one of the top 10 causes of mortality and the first killer among infectious diseases of poverty (IDoPs) worldwide. It disproportionately affects on-third of the world's low-income countries including Ethiopia. One of the factors driving the TB epidemic is the global rise of MDR/XDR-TB and their low detection affect the global TB control progress. Recently, the resistance-associated genetic mutations in MTBC known to confer drug resistance have been detected by rapid molecular diagnostic tests and sequencing methods. In this article, the published literature searched by PubMed database from 2010 to 2021 and English language were considered. The aim of this systematic review was to assess the prevalence of the most common rpoB, katG, and inhA gene mutations associated with multidrug resistance in MTBC clinical strains among TB patients in Ethiopia. Though 22 studies met our eligibility criteria, only 6 studies were included in the final analysis. Using the molecular GenoType MTBDRplus and MTBDRsl line probe assay and sequencing procedures, a total of 932 culture-positive MTBC isolates were examined to determine RIF, INH, and MDR-TB resistance patterns along with rpoB, katG, and inhA gene mutation analysis. As a result, among the genotypically tested MTBC isolates, 119 (12.77%), 83 (8.91%), and 73 (7.32%) isolates were INH, RIF, and MDR-TB resistant, respectively. In any RIF-resistant MTBC strains, the most common single point mutations were in codon 531 (S531L) followed by codon 526 (H526Y) of the rpoB gene. Besides, the most common mutations in any INH-resistant MTBC were strains observed at codon 315 (S315T) and WT probe in the katG gene and at codon C15T and WT1 probe in the inhA promoter region. Detection of resistance allele in rpoB, KatG, and inhA genes for RIF and INH could serve as a marker for MDR-TB strains. Tracking the most common S531L, S315T, and C15T mutations in rpoB, katG, and inhA genes among RIF- and INH-resistant isolates would be valuable in TB diagnostics and treatment regimens, and could reduce the development and risk of MDR/XDR-TB drug-resistance patterns.

Xpert MTB/XDR for detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin.

Xpert MTB/XDR for detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin.

Characterization of rifampicin-resistant Mycobacterium tuberculosis in Khyber Pakhtunkhwa, Pakistan

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is endemic in Pakistan. Resistance to both firstline rifampicin and isoniazid drugs (multidrug-resistant TB; MDR-TB) is hampering disease control. Rifampicin resistance is attributed to rpoB gene mutations, but rpoA and rpoC loci may also be involved. To characterise underlying rifampicin resistance mutations in the TB endemic province of Khyber Pakhtunkhwa, we sequenced 51 M. tuberculosis isolates collected between 2016 and 2019; predominantly, MDR-TB (n = 44; 86.3%) and lineage 3 (n = 30, 58.8%) strains. We found that known mutations in rpoB (e.g. S405L), katG (e.g. S315T), or inhA promoter loci explain the MDR-TB. There were 24 unique mutations in rpoA, rpoB, and rpoC genes, including four previously unreported. Five instances of within-host resistance diversity were observed, where two were a mixture of MDR-TB strains containing mutations in rpoB, katG, and the inhA promoter region, as well as compensatory mutations in rpoC. Heteroresistance was observed in two isolates with a single lineage. Such complexity may reflect the high transmission nature of the Khyber Pakhtunkhwa setting. Our study reinforces the need to apply sequencing approaches to capture the full-extent of MDR-TB genetic diversity, to understand transmission, and to inform TB control activities in the highly endemic setting of Pakistan.

Rapid Molecular Diagnosis of Tuberculosis and Its Resistance to Rifampicin and Isoniazid with Automated MDR/MTB ELITe MGB® Assay.

The MDR/MTB ELITe MGB® kit (ELITech) carried on the ELITe InGenius® platform is a new real-time PCR assay allowing automated extraction and detection of DNA of the Mycobacterium tuberculosis complex (MTB) and mutations in the rpoB and katG genes and inhA promoter region (pro-inhA) associated to resistance to rifampicin and isoniazid, the two markers of multidrug-resistant TB (MDR). We assessed the performances of the test on a collection of strains (n = 54) and a set of clinical samples (n = 242) from routine practice, comparatively to TB diagnosis and genotypic drug susceptibility testing (gDST) as references. Regarding the 242 clinical samples, the sensitivity and specificity of MTB detection by ELITe were 90.9% and 97.5%, respectively. For the detection of resistance-conferring mutations on positive clinical samples, we observed perfect agreement with gDST for katG and pro-inhA (κ = 1.0) and two discordant results for rpoB (κ = 0.82). Considering the 54 cultured strains, very good agreement with gDST was observed for the detection of the 25 distinct mutations in rpoB, katG, and pro-inhA, (κ = 0.95, 0.88, and 0.95, respectively). In conclusion, the automated MDR/MTB ELITe MGB® assay shows great promise and appears to be a valuable tool for rapid detection of pre-MDR- and MDR-TB directly from clinical specimens.

Evaluation of the frequency of mutation genes in multidrug-resistant tuberculosis (MDR-TB) strains in Beijing, China

The aim of this study was to explore the frequency and distribution of gene mutations that are related to isoniazid (INH) and rifampin (RIF)-resistance in the strains of the multidrug-resistant tuberculosis (MDR-TB) Mycobacterium tuberculosis (M.tb) in Beijing, China. In this retrospective study, the genotypes of 173 MDR-TB strains were analysed by spoligotyping. The katG, inhA genes and the promoter region of inhA, in which genetic mutations confer INH resistance; and the rpoB gene, in which genetic mutations confer RIF resistance, were sequenced. The percentage of resistance-associated nucleotide alterations among the strains of different genotypes was also analysed. In total, 90.8% (157/173) of the MDR strains belonged to the Beijing genotype. Population characteristics were not significantly different among the strains of different genotypes. In total, 50.3% (87/173) strains had mutations at codon S315T of katG; 16.8% (29/173) of strains had mutations in the inhA promoter region; of them, 5.5% (15/173) had point mutations at -15 base (C→T) of the inhA promoter region. In total, 86.7% (150/173) strains had mutations at rpoB gene; of them, 40% (69/173) strains had mutations at codon S531L of rpoB. The frequency of mutations was not significantly higher in Beijing genotypic MDR strains than in non-Beijing genotypes. Beijing genotypic MDR-TB strains were spreading in Beijing and present a major challenge to TB control in this region. A high prevalence of katG Ser315Thr, inhA promoter region (-15C→T) and rpoB (S531L) mutations was observed. Molecular diagnostics based on gene mutations was a useful method for rapid detection of MDR-TB in Beijing, China.

Variations in rifampicin and isoniazid resistance associated genetic mutations among drug naïve and recurrence cases of pulmonary tuberculosis

BackgroundThe resistance to first-line drugs can increase the risk of treatment failure and development of resistance to other anti-TB drugs. In TB endemic settings, a considerable rate of recurrence cases exhibited each, year which adds significant burden to the prevalence of disease worldwide. MethodsA total of 562 sputum samples were collected from presumptive positive clinical cases of MDR tuberculosis. Treatment history and demographic data of the patients were obtained after informed consent. Xpert MTB/RIF assay was performed for simultaneous detection of MTB and rifampicin resistance. The mutation patterns of isoniazid and rifampicin were observed after multiplex PCR and reverse hybridization by Genotype® MTBDRplus version 2.0 assay. ResultsA total of 73 of 97 cases (75.2%) of treatment failure were found positive for MDR-TB, whereas 79.6% newly diagnosed and 72.9% default cases were MDR in our isolates. The mutation of rpoB S531L was slightly higher in new treatment cases (89.3%) as compared to the default (80.4%) and failure cases (84.8%), whereas rpoB D516V mutation was more prevalent in default cases (19.6%) with complete absence of rpoB 526 mutation, which was observed in the other two types of cases. The mutation pattern of katG resistance differed among drug naïve and recurrence cases. The resistance in newly diagnosed cases was mostly conferred by katG 315 (49.1%) whereas in default (70.8%) and failure cases (63.3%) isoniazid resistance was commonly associated with katG S315T1 mutation. Mutations in inhA promoter region occurred at nucleotide position −8 and −15. In new cases the rate of mutation of C-15T was 3.7% and T-8A was 1.5% while in treatment failure cases the frequency for C-15T and T-8C was 2.5 and 3.8% respectively. However, no inhA promoter region mediated mutations were detected in default treatment cases. ConclusionRetreated cases are at more risk of developing hot spot mutations. An unusual difference in mutation pattern was determined in naïve and recurrence cases. Some mutations were exclusively associated with the retreatment of 35anti-TB drugs which suggest the increased risk of resistance with poor treatment outcome.

A retrospective cohort study of isoniazid-resistant tuberculosis treatment outcomes and isoniazid resistance-associated mutations in eastern China from 2013 to 2018

ObjectivesThe current situation of isoniazid-resistant, rifampicin-susceptible tuberculosis (Hr-TB) and associated genetic factors is not clear in China. MethodsA retrospective cohort study was conducted from 2013 to 2018 in Jiangsu Province, China. Phenotypic Hr-TB were identified by drug susceptibility testing on Lowenstein–Jensen medium and using a Mycobacterium Growth Indicator Tube 960 (MGIT 960) system, and mutations in the katG 315 codon and inhA promoter nucleotides –8, –15 and –16 were determined by GenoType MTBDRplus and sequencing. All of the Hr-TB patients enrolled were followed up until June 2019. ResultsA total of 1416 smear-positive sputum samples were collected, of which 57 were excluded due to the presence of nontuberculous mycobacteria. Finally, 63/1359 (4.6%) were determined as Hr-TB. After follow-up, 11 Hr-TB patients (17.5%) showed an unfavourable outcome, of whom 5 (7.9%) relapsed, 4 (6.3%) had treatment failure and 2 (3.2%) died. A total of 52 isolates (82.5%) were detected with either katG 315 or inhA promoter nucleotide –8, –15 or –16 mutations, whereas no canonical mutations were found in 8 isolates (12.7%); 3 isolates failed in mutation detection. TB history was found to be associated with unfavourable outcomes for Hr-TB (odds ratio = 6.13, 95% confidence interval 1.05–35.82; P = 0.04). However, mutations in katG 315 and the inhA promoter region were not found to be associated with Hr-TB unfavourable outcomes (P = 0.15). ConclusionUnfavourable outcomes for Hr-TB are serious in eastern China, especially for previously treated patients. Meanwhile, current genetic determination of Hr-TB is inadequate.

ISONIAZID - MONORESISTANT TUBERCULOSIS IN BULGARIA

Background. Isoniazid is a common drug in the treatment regimens for TB infection. Monoisoniazid resistance reduces the probability of a successful treatment outcome and increases the risk of acquiring additional drug resistance.
 Material and methods. For the period 2015-2016 a total of 36 TB cases were confirmed in NRL TB, Sofia, as isoniazid-monoresistant Mycobacterium tuberculosis complex. Minimum inhibitory concentration testing for isoniazid was conducted with BАСТЕС MGIT 960 System in the following concentrations of the drug: 0.1 μg/ml, 0.15μg/ml, 0.2μg/ml, 0.3μg/ml, 0.4μg/ml. Molecular testing was performed with GenoType ® MTBDRplus in order to detect the most common mutations associated with resistance to isoniazid.
 Results. Only 25% of the tested M. tuberculosis complex isolates with phenotypic isoniazid monoresistance had the S315T1 mutation in katG; all isolates were with MIC over 0.4 μg/ml. C15T in the promoter region of inhA was detected in 22.22% of cases and only 1 of them showed MIC below 0.4 μg/ml. No mutations were detected in nearly half of the cases (n=19, 52.78%) and most of these isolates were with lower MIC values (n=12). Conclusion. The rapid testing with GenoType ® MTBDRplus can be used as a screening procedure indicating whether further examination of isoniazid MIC is relevant in resistant M. tuberculosis and whether higher doses could be considered.

Molecular identification of mutations conferring resistance to rifampin, isoniazid and pyrazinamide among Mycobacterium tuberculosis isolates from Iran

Here, we aimed to determine the susceptibility of 70 Mycobacterium tuberculosis isolates obtained from different regions of the country to 8 anti-tuberculosis (anti-TB) drugs and possible underlying mechanisms causing resistance to rifampin, isoniazid, and pyrazinamide. The susceptibility of 70 isolates of M. tuberculosis to anti-TB drugs was tested using proportion method. Strains showing resistance to the first line anti-TB drugs were subjected to PCR amplification and sequencing of the rpoB, katG, ahpC, pncA genes, inhA promoter and oxyR-ahpC intergenic regions to detect resistance conferring mutations. Overall, 77.1% and 77.1% of isolates were resistant to at least one of the tested first- and second-line drugs, respectively. Within the rpoB gene the highest rate of mutation was found in codons 531(450) (56.3%), and 533(452) (12.5%). Also, codons 315 (42.4%) of katG, positions -48, -72 and -77 of oxyR-ahpC (total= 3, 9.1%) and -15 of inhA promoter region (33.3%) were the most altered positions in isoniazid resistant isolates. Only a single mutation was detected for pncA among resistant isolates. High prevalence of resistance to essential anti-TB drugs among M. tuberculosis strains isolated from retreated tuberculosis cases is alarming issue necessitating immediate action to prevent the spread of drug resistant isolates in the country.

Prevalence and genetic profiles of isoniazid resistance in tuberculosis patients: A multicountry analysis of cross-sectional data.

BackgroundThe surveillance of drug resistance among tuberculosis (TB) patients is central to combatting the global TB epidemic and preventing the spread of antimicrobial resistance. Isoniazid and rifampicin are two of the most powerful first-line anti-TB medicines, and resistance to either of them increases the risk of treatment failure, relapse, or acquisition of resistance to other drugs. The global prevalence of rifampicin resistance is well documented, occurring in 3.4% (95% CI 2.5%–4.4%) of new TB patients and 18% (95% CI 7.6%–31%) of previously treated TB patients in 2018, whereas the prevalence of isoniazid resistance at global and regional levels is less understood. In 2018, the World Health Organization (WHO) recommended a modified 6-month treatment regimen for people with isoniazid-resistant, rifampicin-susceptible TB (Hr-TB), which includes rifampicin, pyrazinamide, ethambutol, and levofloxacin. We estimated the global prevalence of Hr-TB among TB patients and investigated associated phenotypic and genotypic drug resistance patterns.Methods and findingsAggregated drug resistance data reported to WHO from either routine continuous surveillance or nationally representative periodic surveys of TB patients for the period 2003–2017 were reviewed. Isoniazid data were available from 156 countries or territories for 211,753 patients. Among these, the global prevalence of Hr-TB was 7.4% (95% CI 6.5%–8.4%) among new TB patients and 11.4% (95% CI 9.4%–13.4%) among previously treated TB patients. Additional data on pyrazinamide and levofloxacin resistance were available from 6 countries (Azerbaijan, Bangladesh, Belarus, Pakistan, the Philippines, and South Africa). There were no cases of resistance to both pyrazinamide and levofloxacin among Hr-TB patients, except for the Philippines (1.8%, 95% CI 0.2–6.4) and Belarus (5.3%, 95% CI 0.1–26.0). Sequencing data for all genomic regions involved in isoniazid resistance were available for 4,563 patients. Among the 1,174 isolates that were resistant by either phenotypic testing or sequencing, 78.6% (95% CI 76.1%–80.9%) had resistance-conferring mutations in the katG gene and 14.6% (95% CI 12.7%–16.8%) in both katG and the inhA promoter region. For 6.8% (95% CI 5.4%–8.4%) of patients, mutations occurred in the inhA promoter alone, for whom an increased dose of isoniazid may be considered. The main limitations of this study are that most analyses were performed at the national rather than individual patient level and that the quality of laboratory testing may vary between countries.ConclusionsIn this study, the prevalence of Hr-TB among TB patients was higher than the prevalence of rifampicin resistance globally. Many patients with Hr-TB would be missed by current diagnostic algorithms driven by rifampicin testing, highlighting the need for new rapid molecular technologies to ensure access to appropriate treatment and care. The low prevalence of resistance to pyrazinamide and fluoroquinolones among patients with Hr-TB provides further justification for the recommended modified treatment regimen.

Evaluation of GenoType MTBDRplus for the detection of drug-resistant Mycobacterium tuberculosis on isolates from Karachi, Pakistan.

ObjectiveTo compare the diagnostic performance of the GenoType MRBDRplus assay with the gold standard phenotypic drug susceptibility testing in the detection of drug resistance among culture isolates obtained from patients in Karachi, Pakistan.DesignMycobacterium tuberculosis isolates were obtained from 96 consecutive tuberculosis patients found to have resistance to isoniazid from two health centers in Karachi (January-November 2017). Isolates were tested for drug resistance against rifampin and isoniazid using the MTBDRplus assay. Results were compared with conventional drug-susceptibility testing and the frequency of specific mutations were reported.ResultsThe MTBDRplus assay had a sensitivity for rifampin resistance of 98.8% (95% CI: 93.4–100) and for isoniazid resistance of 90.6% (95% CI: 83.0–95.6). The MTBDRplus assay showed mutations in rpoB in 81 of the 96 (84.4%) isolates. Of the 87 isolates showing resistance to isoniazid via the MTBDRplus assay, 71 (74.0%) isolates had mutations in the katG gene only, 15 (15.6%) isolates had mutations in the inhA promoter region, and 1 (1.0%) showed mutations in both genes.ConclusionThe GenoType MTBDRplus assay in Pakistan can identify subgroups at high-risk of having isolates with mutations in the katG and/or inhA genes. Understanding the local burden of these mutations have implications for local diagnostic and treatment guidelines.

Resistencia cruzada entre isoniacida y etionamida y su alta correlación con la mutación C-15T en aislamientos de Mycobacterium tuberculosis de Perú

Several studies have shown cross-resistance between isoniazid and ethionamide, 2of the drugs used in the treatment of multidrug-resistant tuberculosis. The objective of this study was to determine the cross-resistance between both drugs in Mycobacterium tuberculosis isolates from a hospital with high incidence of tuberculosis in Lima, Peru. The frequency of mutations to isoniazid in the katG gene and the inhA promoter region was identified by the Genotype MTBDRplus v2.0 molecular test. The gold standard Agar Proportion method (APM) allowed todetect resistance to isoniazid and ethionamide. Of 107 isoniazid-resistant isolates (54 multidrug-resistant isolates identified by the Genotype MTBDRplus test, 45.8% (49/107) were also resistant to ethionamide by the APM. Mutations were found in the katG gene in 50.5% (54/107), in the promoter region inhA in 23.3% (25/107) and 14.0% (15/107) that share both mutations in the resistant isolates to INH. The absence of the wild type and mutation bands indicated that 12.1% (13/107) of the isolates were resistant to INH. The mutation C-15T in the inhA promoter region showed a strong association with resistance to ethionamide in 73.4% (36/49) of the isolates analyzed. The results of the present study suggest that the identification of mutations related to resistance to isoniazid, especially in the inhA promoter region, could be very useful to identify cross-resistance to ethionamide and improve the treatment of individuals suffering from this disease.

DESAIN PRIMER UNTUK AMPLIFIKASI REGIO PROMOTER GEN inhA ISOLAT P016 MULTIDRUG RESISTANCE Mycobacterium tuberculosis DENGAN METODE POLYMERASE CHAIN REACTION

Mutations in the inhA promoter region are responsible for isoniazid resistance and cross-resistance to ethionamide. To identify mutations with polymerase chain reaction (PCR), a pair of primers that can amplify the target region. This aim of this study was to obtain the best primer pair to amplify the inhA promoter region using the Clone Manager Suite 6 program. The inhA promoter sequence (Genbank: U66801) obtained from the www.ncbi.nlm.nih.gov was used as a template. The design results obtained the best primer pair tested in vitro using Polymerase Chain Reaction (PCR) method. The PCR amplification process was performed for 40 cycles with the following conditions: predenaturase (95oC for 15 minutes), denaturation (94 oC for 1 minute), annealing (56 oC for 1 minute 30 seconds), elongation (72 oC for 2 minutes), and final elongation (72 oC for 10 minutes). Detection of PCR products was performed in agarose gel electrophoresis 1.3% w / v and visualized by UV transluminator tool. The results obtained were forward primers of 5'-GGTCGAAGTGTGCTGAGTC-3 'and reverse primer 5'-TGCTCTTCTACCGCCGTGA-3' which met the good primary criterion based on Clone Manager Suite 6. The pair of primers has been able to amplify the inhA promoter region by the length of product produced at 373 bp.

Molecular diagnosis of multidrug-resistant tuberculosis in sputum samples by melting curve analysis

To analyze melting curves for the diagnosis of multidrug-resistant tuberculosis from sputum samples. Sputum samples (n = 250) were collected from patients with clinical suspicion of pulmonary tuberculosis as a result of bacilloscopy and cultured in solid medium Lowenstein Jensen. According to the reference method, 124 samples sensitive to rifampicin and isoniazid, 24 resistant to rifampicin, 33 resistant to isoniazid, and 69 multidrug-resistant were used. It was evaluated by real-time PCR and then by melting curves, the rpoB gene was used as a biomarker of rifampicin resistance, and the katG gene and inhA promoter region were used as biomarkers of isoniazid resistance. The H37Rv strain was considered a drug-sensitive control. The results of the reference method and the results of the melting curve analysis were compared to evaluate the parameters of sensitivity, specificity, positive predictive value and negative predictive value. Rifampicin resistance showed a sensitivity of 90.3%, specificity of 90.4%, positive predictive value of 84.8% and negative predictive value of 94.0%. Isoniazid resistance showed a sensitivity of 90.2%, specificity of 93.9%, positive predictive value of 91.1% and negative predictive value of 93.3%. The detection of multidrug-resistant tuberculosis showed values of 89.9%, 90.6%, 78.5% and 95.9% for sensitivity, specificity, positive predictive value and negative predictive value, respectively. The melting curve analysis showed to be safe and reliable to be used in the rapid diagnosis of multidrug-resistant tuberculosis in sputum samples.

Prevalence of mutations in genes associated with isoniazid resistance in Mycobacterium tuberculosis isolates from re-treated smear-positive pulmonary tuberculosis patients: A meta-analysis.

The prevalence of isoniazid (INH) monoresistance is high in India. In this study, molecular epidemiological characteristics associated with INH resistance mutations in Mycobacterium tuberculosis in codon katG315 and in the promoter region of the inhA gene were investigated. Sputum specimens of smear-positive tuberculosis (TB) patients were subjected to GenoType MTBDRplus assay to identify katG and inhA mutations in M. tuberculosis. In addition to the current study, 17 publications assessed 14100 genotypically resistant M. tuberculosis isolates for mutations in katG inclusive of codon 315. In total, 1821 (11.8%) of 15438 INH-resistant strains had detectable mutations: 71.0% in katG315 and 29.0% in the inhA promoter region. The prevalence of IHN monoresistance was 89.1% in the economically-active age group, 0.4% in the paediatric age group and 10.5% in those aged >60years; the rate in males and females was 12.0% and 10.8%, respectively. Meta-analysis derived a pooled katGS315T resistant TB prevalence of 67.3% (95% CI 59.3-75.4%) with Q=732.19, I2=98.35% and P=0.000 for treated TB cases. INH resistance was spread widely and transmission of INH-resistant isolates, especially with katG315T mutation, was confirmed. Therefore, it is important to diagnose katG315T mutants among INH-resistant strains as it may be a risk factor for subsequent development of multidrug-resistant TB (MDR-TB). Prompt detection of patients with INH-resistant strains would expedite modification of treatment regimens, and appropriate infection control measures could be taken in time to diminish the risk of further development and transmission of MDR-TB.

The occurrence and frequency of genomic mutations that mediate Isoniazid and Rifampicin resistance in Mycobacterium tuberculosis isolates from untreated pulmonary Tuberculosis cases in urban Blantyre, Malawi.

The emergence and spread of drug-resistant Tuberculosis (TB) is a major public health threat. TB resistance originates in the course of treatment due to genomic mutations in Mycobacterium tuberculosis (MTB). An increase in new cases with drug-resistant TB could be an indicator of high levels of circulating resistant strains. This study was conducted to determine the occurrence and frequency of genomic mutations that mediate Isoniazid (INH) and Rifampicin (RIF) resistance among isolates from untreated TB cases in urban Blantyre, Malawi. A cross-sectional retrospective study was conducted on a panel of 141(n=141) MTB clinical isolates recovered between June 2010 and January 2012 from >2+ Ziehl-Neelsen smear positive new pulmonary-TB patients with no history of treatment. Frozen isolates were revived using the BACTEC MGIT detection system. DNA was extracted using GenoLyse DNA extraction kit and detection of genomic mutations was carried out using the GenoType MTBDRplus Ver 2.0 assay. Out of the 141 isolates studied, 3 (2.1%) were found carrying mutations in the katG gene that confer resistance to Isoniazid (INH). No mutations were detected in the inhA promoter region gene that confer weak INH resistance or in the rpoB gene that confer Rifampicin resistance. All katG mutant genes had a S315T1 single point mutation, a genomic alteration that mediates high INH resistance. The katG mutant gene conferring resistance to INH was the only genomic mutation observed among the isolates studied and the frequency of occurrence was low. Our findings suggest low levels of circulating drug-resistant MTB strains in urban Blantyre, Malawi.

Effective pragmatic approach of diagnosis of multidrug-resistant tuberculosis by high-resolution melt curve assay.

Effective management of multidrug-resistant tuberculosis (MDR-TB) requires cost-effective and rapid screening of rifampicin (RIF) and isoniazid (INH) resistance. Accordingly, a highly promising high-resolution melting (HRM) analysis was evaluated in the detection of mutation in rpoB, katG gene and inhA promoter region in Mycobacterium tuberculosis isolates. A total of 143 M. tuberculosis isolates comprising phenotypically confirmed 94 MDR and 49 sensitive isolates were analyzed by HRM following real-time-polymerase chain reaction in comparison to gold standard of targeted DNA sequencing of rpoB, katG gene and inhA promoter region. HRM correctly identified MDR-TB by rapid and accurate detection of predominantly and infrequently occurring specific single nucleotide polymorphism in rpoB, katG gene and inhA promoter region. rpoB HRM showed sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 98% each respectively. Predominantly, S531 L/W (TCG → TTG/TGG) mutation accounted for 68.47% of RIF resistance followed by H526Y/R (13.04%, CAC → TAC/CGC), D516Y/V/G (10.86%, GAC → TAC/GTC/GGC), Q513P (4.34%, CAA → CCA), and one rare mutation at codon position L533A (CTG → CGG). Combined KatG and inhA HRM sensitivity, specificity, PPV, and NPV were 90%, 100%, 100%, and 84.48% respectively and detected frequent mutation at codon position S315T/I/N (70%, AGC → ACC, AGC → ACT, AGC → AAC) and rare mutation at codon position T314P (3.3%, ACC → CCC) and 329 (2.2%, GAC → GCC) of katG gene. In inhA, mutations were recorded at mostly promoter position - 15 (10%, C → T) and infrequently at - 8 (3.3%, T → G, T → C). HRM assay limitation noticed in recognizing silent mutation in rpoB as a mutant, nondetection of infrequent mutation S310A in katG, and the inability of detecting mutation outside the targeted region of investigated genes. HRM may prove to be a vital molecular assay in rapid screening of TB cases for early detection of MDR TB, leading to early evidenced-based initiation of antitubercular treatment that will significantly reduce MDR transmission.

Variants of katG, inhA and nat genes are not associated with mutations in efflux pump genes (mmpL3 and mmpL7) in isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from India

To understand the impact of efflux pump genes such as mmpL3 and mmpL7 on isoniazid (INH) resistance and to correlate with presence or absence of mutations in essential genes of INH resistance (katG, inhA, and nat) in clinical isolates of Mycobacterium tuberculosis (M. tuberculosis). One hundred (75 resistant and 25 sensitive) clinical isolates of M. tuberculosis from India were selected for the study. The presence of mutations in specific regions of katG, inhA, and nat, efflux pump genes (mmpL3 and mmpL7) associated with INH resistance were analyzed using multiplex allele-specific polymerase chain reaction (MAS-PCR) and DNA sequencing methods, respectively. Substitution mutation AGC-ACC at codon 315 of the katG gene was detected in 65% of resistant isolates. Mutation (C-T at nucleotide position 15) in the inhA promoter region was seen in 22% of resistant isolates. Silent mutation (GGA to GGG) at codon 207 in the nat gene was found in three resistant isolates. No mutations were found in either of the efflux genes (mmpL3 and mmpL7) in any of the isolates. Of the 75 resistant isolates analyzed, 74% had mutation in katG and inhA genes. Thus, this report suggests that the role of mmpL3, mmpL7 and nat genes in INH resistance should not be overestimated in comparison to the primary contribution by katG and inhA in clinical isolates of M. tuberculosis. Further, this concise report is the first of its kind to our knowledge, to show the influence of efflux genes on INH resistance in relation to katG and inhA in clinical isolates of M. tuberculosis.

In Silico Prediction of Antibiotic Resistance in Mycobacterium ulcerans Agy99 through Whole Genome Sequence Analysis.

Buruli ulcer is an emerging infectious disease caused by Mycobacterium ulcerans that has been reported from 33 countries. Antimicrobial agents either alone or in combination with surgery have been proved to be clinically relevant and therapeutic strategies have been deduced mainly from the empirical experience. The genome sequences of M. ulcerans strain AGY99, M. ulcerans ecovar liflandii, and three Mycobacterium marinum strains were analyzed to predict resistance in these bacteria. Fourteen putative antibiotic resistance genes from different antibiotics classes were predicted in M. ulcerans and mutation in katG (R431G) and pncA (T47A, V125I) genes were detected, that confer resistance to isoniazid and pyrazinamide, respectively. No mutations were detected in rpoB, gyrA, gyrB, rpsL, rrs, emb, ethA, 23S ribosomal RNA genes and promoter region of inhA and ahpC genes associated with resistance. Our results reemphasize the usefulness of in silico analysis for the prediction of antibiotic resistance in fastidious bacteria.