Mutations In rpoB Gene Research Articles

Automatic Identification of Individual rpoB Gene Mutations Responsible for Rifampin Resistance in Mycobacterium tuberculosis by Use of Melting Temperature Signatures Generated by the Xpert MTB/RIF Ultra Assay.

Molecular surveillance of rifampin-resistant Mycobacterium tuberculosis can help to monitor the transmission of the disease. The Xpert MTB/RIF Ultra assay detects mutations in the rifampin resistance-determining region (RRDR) of the rpoB gene by the use of melting temperature (Tm ) information from 4 rpoB probes which can fall in one of the 9 different assay-specified Tm windows. The large amount of Tm data generated by the assay offers the possibility of an RRDR genotyping approach more accessible than whole-genome sequencing. In this study, we developed an automated algorithm to specifically identify a wide range of mutations in the rpoB RRDR by utilizing the pattern of the Tm of the 4 probes within the 9 windows generated by the Ultra assay. The algorithm builds a RRDR mutation-specific "Tm signature" reference library from a set of known mutations and then identifies the RRDR genotype of an unknown sample by measuring the Tm distances between the test sample and the reference Tm values. Validated using a set of clinical isolates, the algorithm correctly identified RRDR genotypes of 93% samples with a wide range of rpoB single and double mutations. Our analytical approach showed a great potential for fast RRDR mutation identification and may also be used as a stand-alone method for ruling out relapse or transmission between patients. The algorithm can be further modified and optimized for higher accuracy as more Ultra data become available.

The Application of Xpert Mycobacterium tuberculosis/Rifampicin, Quantitative Polymerase Chain Reaction and High Resolution Melting Curve in the Diagnosis of Superficial Lymph Node TB.

The diagnostic sensitivity and specificity of conventional methods for superficial lymph node tuberculosis (LNTB) are not ideal. We evaluated several novel methods including Xpert Mycobacterium tuberculosis/rifampicin (Xpert MTB/RIF) technology, quantitative fluorescence Polymerase Chain Reaction (qPCR) and High-Resolution Melting Curve (HRMC) in the diagnosis of superficial lymph node TB. Specimens from eighty-one consecutive patients with suspected LNTB and thirteen cases with other lymph node disease were analyzed by Xpert MTB/RIF, qPCR, and HRMC. Among 81 patients with clinical suspicion of LNTB, there were 74 (91.4%) cases positive Mycobacterium tuberculosis Complex (MTBC) of Xpert MTB/RIF, 60 (74%) positive of qPCR, 24 (29.6%) of positive of BACTEC MGIT960 culture, and 13 (16%) cases positive of Roche culture. 38 cases (46.9%) were diagnosed with LNTB. All test methods showed a diagnostic specificity of 100% for LNTB. The sensitivity of molecular biology techniques was significantly higher than that of the traditional diagnostic methods, and Xpert MTB/RIF was the most sensitive diagnostic assay. On Rifampinresistant detection, Xpert MTB/RIF detected three cases (3.7%) with rpoB gene mutation, and Mycobacterium tuberculosis susceptibility testing detected 2 rifampicin-resistant cases (2.4%) which were consistent with Xpert MTB/RIF results. In the Isoniazid-resistant, 7 cases (8.1) of isoniazid resistance mutations (8.1%) were detected by HNC and 1 case was confirmed by Isoniazid susceptibility test. Molecular detection increased the diagnostic sensitivity of LNTB and improved the detection sensitivity for rifampin and isoniazid resistance strain.

MYCOBACTERIUM TUBERCULOSIS;

Objectives: The primary objective of this study is to determine the frequency of rpoB gene mutations within 81-bp RRDR in isolates from Quetta, Pakistan using GeneXpert® MTB/RIF assay. Background: Multi drug resistance is one of the major obstacles in the control of tuberculosis throughout the globe. Study Design: A cross sectional experimental study was designed. Setting: A total of 500 clinical specimens obtained from suspected TB patients at Provincial TB Reference Laboratory, Fatima Jinnah General and Chest Hospital Quetta, Pakistan Period: From January to July 2017. Methodology: Were analyzed by GeneXpert® MTB/RIF assay. Statistical analysis of the data was performed using SPSS version 20. Results: Out of total 500 samples, MTB was detected in 211 (42%) cases by GeneXpert® MTB/RIF assay [positive pulmonary cases 48.8% (206/422) and extra-pulmonary 6.4% (5/78)]. Among 211 MTB positive cases, the assay detected 11 (5.2%) cases with RIF-resistance caused by various rpoB gene mutations within 81-bp RRDR. All the eleven RIF-resistant isolates were found to have mutations only in Probe E and none of cases had RIF-resistance associated with probes A, B, C, and D. Out of 11 RRD cases, 4 (2%) were males and 7 (3.3%) were females. New TB cases were 3 (1.4%) and previously treated cases were 8 (3.8%). Conclusion: In our settings 11/211 (5.2%) of the TB patients showed rifampicin resistance. Probe E mutations (also called codons 531 and 533) were the only rpoB gene mutations detected by GeneXpert® MTB/RIF assay. No mutations were detected in the codons 511, 513, 516, 518, 522 and 526 sequences.

Rapid Detection of Mycobacterium Tuberculosis in Lung Tissue and Analysis of Drug Resistance

This study investigated the methods for optimizing the workflow for improving the diagnostic efficiency for detection of Mycobacterium tuberculosis (MTB) in lung tissue specimens. A total of 278 specimens were used in this study. M. tuberculosis in fresh lung tissue samples was detected by BACTEC MGIT 960 culture system, culturing L-form MTB, rifampicin (RFP) and levofloxacin (LVFX) susceptibility test, and ribonucleic acid (RNA) simultaneous amplification and testing (SAT). Specimen samples were embedded in paraffin and serially sectioned. The sections were subjected to Ziehl-Neelsen staining and Intensified Kinyoun (IK) acid-fast staining. The suspected MTB or L-form MTB specimens were further investigated by deoxyribonucleic acid (DNA) sequencing and fluorescence polymerase chain reaction (PCR) melting curve method to detect the mutations in rpoB gene and gyrA gene. Thirteen specimens were suspected as MTB positive, and 37 specimens were suspected to be L-form MTB positive by Ziehl-Neelsen staining and IK acid-fast staining. Among the 50 specimens, the number of MTB positive specimens detected by SAT, DNA sequencing, and fluorescence PCR melting curve method was 43, 44, and 45, respectively. Only 11 MTB positive specimens were detected by BACTEC MGIT 960 culture system or by culturing L-form MTB. Mutations detected in rpoB gene and gyrA gene by fluorescence PCR melting curve method were similar to those detected by DNA sequencing. Some specimens, detected by melting curve method, exhibited varied drug resistance to RFP, suggesting heterogeneous resistance. Among the remaining 228 specimens, there was no MTB or L-form MTB detected by BACTEC MGIT 960 culture system. However, 5 specimens were detected to be MTB positive by the SAT method. The fluorescent PCR melting curve method, which has a high level of automation and high sensitivity and specificity, could effectively detect heterozygous drug resistance of MTB in lung tissue samples, which is important for clinicians to effectively formulate a therapeutic strategy.

Sequencing of the entire rpob gene and characterization of mutations in isolates of Mycobacterium tuberculosis circulating in an endemic tuberculosis setting.

To evaluate the use of a sequencing procedure for the entire rpoB gene of Mycobacterium tuberculosis to identify mutations pre-rifampicin resistance determining region (RRDR), within RRDR, and post-RRDR in isolates circulating in a region affected by tuberculosis (TB). Five primers were designed, with which five DNA fragments of rpoB were obtained, sequenced by Sanger, and analysed in silico in order to identify mutations over the entire rpoB gene in rifampicin-sensitive and rifampicin-resistant TB. It was possible to analyse the entire rpoB gene in five rifampicin-sensitive and 15 rifampicin-resistant isolates. Thirty-six mutations were identified. Two mutations were found pre-RRDR, nine within-RRDR and 25 post-RRDR. The most frequent mutations within RRDR were S531L (53%), followed by S512T (20%), all of which were found in rifampicin-resistant isolates. Of the 25 mutations found post-RRDR, 14 were only in resistant isolates, and the most frequent was D853N, which was present in 85% of isolates. Mutations E818K, D836N and T882P were observed in 80% of the rifampicin-resistant and rifampicin-sensitive isolates. The proposed sequencing method allowed identification of mutations in the entire rpoB gene. This procedure represents a useful tool for diagnosing rifampicin resistance. The number of mutations that were found raises new questions about the diversity of mutations in the rpoB gene and their role in rifampicin resistance in regions where TB is endemic.

Detection of Ser450Leu mutation in rpoB gene of Mycobacterium tuberculosis by allele-specific loop-mediated isothermal DNA amplification method

To identify genetic mutations a rather time-consuming and expensive method of polymerase chain reaction (PCR) is widely used. The aim of the present work was to evaluate the possibility of using the two schemes of the method of allele-specific isothermal loop amplification (LAMP) to detect the TCG/TTG (S450L) mutation in the rpoB gene of Mycobacterium tuberculosis. 48 clinical isolates of M. tuberculosis and 11 samples of sputum were used, randomized and obtained in the microbiological laboratory of the city of Novosibirsk from incident patients. It is shown that the use of an analysis scheme using the allele-specific primer FIP compared to F3 has the best resolution: the difference between the amplification time of the mutation and the wild type allele was 22 ± 2,4 versus 13 ± 4,1 minutes (p = 0,0011). When using 100 DNA genomic equivalents a true positive signal (amplification of the rpoB gene with a mutation using the corresponding allele-specific primer) was detected after 29,4 ± 3,4 minutes. A positive signal was visualized after adding SYBR Green I to the reaction, both when illuminated with daylight and when using a UV transilluminator. Using the developed method the DNA sample of 20 RIFR isolates from M. tuberculosis was analyzed containing the Ser450Leu mutation in the rpoB gene, 10 RIFR isolates containing other mutations in the rpoB gene and 18 RIFs isolates without any mutations; the presence of mutations in the samples was determined using classical Sanger sequencing. The sensitivity and specificity of LAMP for detecting a Ser450Leu mutation in the rpoB gene was 100%. This approach allows the use of crude lysates of mycobacteria as DNA, which reduces the total analysis time to 1,5 hour.

РАСПРОСТРАНЕННОСТЬ МУТАЦИЙ В ГЕНАХ МИКОБАКТЕРИЙ ТУБЕРКУЛЕЗА, КОДИРУЮЩИХ ЛЕКАРСТВЕННУЮ УСТОЙЧИВОСТЬ К ИЗОНИАЗИДУ И РИФАМПИЦИНУ, У БОЛЬНЫХ ТУБЕРКУЛЕЗОМ В РАЗНЫХ ВОЗРАСТНЫХ ГРУППАХ

The objective : to study the prevalence and ranges of mutations in katG, inhA, and ahpC genes coding resistance to isoniazid, and in rpoB gene coding resistance to rifampicin in patients with pulmonary tuberculosis from different age groups. Subjects and methods . 253 patients were examined; based on their age they were divided into 3 groups (Group 1 – from 18 to 30 years, Group 2 – from 31 to 60 years, group 3 – from 61 to 80 years). Biological microchips with TB-Biochip reagent kit (Russia) were used. Results . In Groups 1 and 2, compared to Group 3, mutations were recorded more often simultaneously in katG, inhA, ahpC genes and rpoB genes which meant that multiple drug resistance (MDR) prevailed (32.4 and 36.1% against 13.2%, respectively). In general, mutations in katG gene (including MDR + isolated isoniazid resistance) were also confidently more frequent in Groups 1 and 2 (30.9 and 32.7%) versus Group 3 (10.5%). Similar changes were found in the prevalence of the adverse type of ser 315-> Thr mutation of katG gene, which was registered in 23.5% of cases in Group 1, in 21.8% of cases in Group 2, and in 7.9% – in Group 3. In Group 3, mutations in rpoB gene (including MDR) were less frequently recorded and made 26.5% versus 45.5% in Group 1 and 46.9% in Group 2. There were no confident differences in ranges of mutations in rpoB gene between the examined groups of patients. Thus, a significantly lower level of resistance to isoniazid and rifampicin in elderly and senile tuberculosis patients was found compared to young and middle-aged patients, which was registered at the level of genetic mutations.

Conventional and molecular determination of drug resistance in Mycobacterium tuberculosis and Mycobacterium bovis isolates in cattle

This study was aimed out to explore the presence of drug resistance among M. tuberculosis and M. bovis isolates (n = 51) of bovine origin by conventional broth microdilution method and molecular methods. By broth microdilution method, 16 isolates were found to be resistant to isoniazid, 08 isolates were resistant to pyrazinamide, 09 isolates were resistant to rifampicin and 07 isolates were found to be resistant for ethambutol. Two isolates showed resistance to rifampicin and pyrazinamide, one isolate showed resistance to pyrazinamide and ethambutol and 03 isolates showed resistance to isoniazid and ethambutol. None of isolates showed multi drug resistance (MDR). Other than the M. bovis strains, none of the other M. tuberculosis isolates showed any resistance to pyrazinamide. Molecular methods by multiplex PCR targeting katG, pncA, rpoB genes, multiplex allele specific PCR to detect mutation in embB codon 306 and sequencing showed point mutation in katG and rpoB gene. No mutation could be detected in the embB gene by multiplex allele specific PCR. The results indicates that further elaborate studies need to be carried out due to the presence of drug resistant M. tuberculosis in bovines which could be due to spill over from human in tuberculosis endemic areas making TB eradication programme more challenging.

Evaluation of the expression of cytokines and chemokines in macrophages in response to rifampin-monoresistant Mycobacterium tuberculosis and H37Rv strain

Macrophages are the primary phagocytes in the lungs and a part of the host defense system against Mycobacterium tuberculosis (Mtb), involved in the primary immune response. While several studies have assessed the effects of resistance to rifampin on Mtb physiology, the consequences of mutations in genes encoding the beta subunit of RNA polymerase (rpoB) for host-pathogen interactions remain poorly understood. In this study, rifampin-monoresistant (RMR) Mtb and H37Rv strains were used to infect the THP-1-derived macrophages. Real-time quantitative reverse transcription PCR assay was carried out to determine mRNA expression in 84 cytokine and chemokine genes. Production of specific cytokines and chemokines was measured by ELISA assay. In conclusion, the current study shed more light on the fitness cost of RMR strain and the potential effects of rpoB gene mutations on Mtb-host interactions. These results initially demonstrate that the Mtb carrying the rpoB-S450L can modulate macrophage responses to mediate bacterial survival.

Molecular detection of rifampin, isoniazid, and ofloxacin resistance in Iranian isolates of <em>Mycobacterium tuberculosis</em> by high-resolution melting analysis

BackgroundThe emergence of drug resistance among Mycobacterium tuberculosis (MTB) strains is a serious health concern worldwide. The development of rapid molecular diagnostic methods in recent years has a significant impact on the early detection of resistance to major anti-TB drugs in MTB isolates, which helps in employing appropriate treatment regimen and prevents the spread of drug-resistant strains. This study was designed to evaluate the efficacy of real-time PCR and high-resolution melting (HRM) curve analysis for the determination of resistance to rifampin (RIF), isoniazid (INH), and ofloxacin (OFX) in MTB isolates and to investigate their resistance-related mutations.MethodsHRM analysis was performed to screen 52 (32 drug-resistant and 20 fully susceptible) MTB clinical isolates for mutations in rpoB, katG, mab-inhA, and gyrA genes. The HRM results were then confirmed by DNA sequencing.ResultsIn total, 32 phenotypically resistant isolates, comprising 18 RIF-, 16 INH-, and five OFX- resistant strains, were investigated. HRM analysis successfully identified 15 out of 18 mutations in rpoB, 14 out of 16 mutations in katG and mab-inhA, and four out of five mutations in gyrA conferring resistance to RIF, INH, and OFX, respectively. The obtained sensitivity and specificity, respectively, for HRM in comparison with phenotypic susceptibility testing were found to be 83.3% and 100% for RIF, 87.5% and 100% for INH, and 80% and 100% for OFX. In five resistant strains (12.8%), no mutation was detected by using HRM and DNA sequencing.ConclusionHRM assay is a rapid, accurate, and cost-effective method possessing high sensitivity and specificity for the determination of antibiotic resistance among MTB clinical isolates and screening of their associated mutations. This method can generate results in a shorter period of time than taken by the phenotypic susceptibility testing and also allows for timely treatment and prevention of the emergence of possible MDR strains.

Phenotypic and genotypic drug sensitivity of <em>Mycobacterium tuberculosis</em> complex isolated from South Omo Zone, Southern Ethiopia

BackgroundKnowledge of drug-sensitivity patterns of Mycobacterium tuberculosis complex (MTBC) strains isolated from patients is an important aspect of TB control strategy. This study was conducted to evaluate the drug sensitivity of MTBC isolates in South Omo, southern Ethiopia.Materials and methodsA total of 161 MTBC isolates (153 from new cases and eight re-treatment TB cases) were isolated using Lowenstein Jensen medium of which 126 isolates were able to be tested for drug sensitivity by BACTEC™MGIT™ 960 system, while all the 161 isolates were tested by GenoType® MTBDRplus VER 2.0. Descriptive statistics and logistic regression were used to express and present results.ResultsOn the basis of MGIT 960 system, the prevalence of mono-resistance was 9.2% (11/119) in the new cases, although neither poly-resistance nor multidrug resistance (MDR) was recorded in these cases. On the basis of GenoType MTBDRplus assay, two of the 153 isolates (1.3%) of the new cases were mono-resistant for rifampicin (RIF) and one of these isolates had known rpoB gene mutation (H526D). One of the eight (12.5%) isolates obtained from the re-treatment cases was MDR with rpoB gene mutation (D516V) and katG gene mutation (S315T2). Taking MGIT 960 system as a gold standard, the sensitivities of the MTBDRplus assay were 33.3%, 100% and 100% for detection of resistance to isoniazid, RIF and MDR, respectively. On the other hand, its specificities were 99.2%, 100% and 100% for detection of resistance to RIF, isoniazid and MDR, respectively.ConclusionThe magnitude of drug resistance was relatively low in the new TB cases of South Omo as compared to the reports from the other regions of the country. This is encouraging and hence the TB Control Program in the Zone should strengthen its program so that the emergence of drug resistance is inhibited.

Mutations of rpob Gene Associated with Rifampin Resistance among Mycobacterium Tuberculosis Isolated in Tuberculosis Regional Reference Laboratory in Northeast of Iran during 2015-2016.

Drug resistance is a leading concern in control of TB. Resistance against rifampin as one of the most important drugs in the treatment of Mycobacterium tuberculosis is caused by mutations in the 81-base pair region of the rpoB gene encoding the β-subunit of RNA polymerase. This study aimed to characterize the mutations in the rpoB gene associated with rifampin resistance among M. tuberculosis. This study was conducted on referred samples of patients who did not respond to anti-TB treatment, in Tuberculosis Regional Reference Laboratory at Shariati Hospital. Drug susceptibility of M. tuberculosis isolates was surveyed using a proportional method on LJ medium. The isolates with resistant to rifampin were reconfirmed and then the rpoB gene was amplified and sequenced. Among 27 resistant cases, 8, 11 and 8 people were from Iran, Afghanistan, and Turkmenistan, respectively. In 26 out of 27 isolates, rpoB gene mutations were observed. The most prevalent mutations belonged to the codon 53. The most prevalent mutations belonged to the TCG (Ser) 531TTG (leu) with prevalence 51.8% (n=14), and GAC (Asp)516TAC (Tyr), CAC (His) 526GAC (Asp) and CAC (His) 526TAC(Tyr) mutations with prevalence 14.8%(n=4). Twenty-three isolates had just one mutation. The use of rpoB gene sequencing led to the lack of the need for growth of the organism in the culture medium, the direct use of clinical samples, reduction of biological risks and a detection about 96.3% of MDR TB cases lowering the cost of the treatment.

Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis.

Rifampicin (RIF) plays a pivotal role in the treatment of tuberculosis due to its bactericidal effects. Because the action of RIF is on rpoB gene encoding RNA polymerase β subunit, 95% of RIF resistant mutations are present in rpoB gene. The majority of the mutations in rpoB gene are found within an 81bp RIF-resistance determining region (RRDR). Literatures on RIF resistant mutations published between 2010 and 2016 were thoroughly reviewed. The most commonly mutated codons in RRDR of rpoB gene are 531, 526 and 516. The possibilities of absence of mutation in RRDR of rpoB gene in MDR-TB isolates in few studies was due to existence of other rare rpoB mutations outside RRDR or different mechanism of rifampicin resistance. Molecular methods which can identify extensive mutations associated with multiple anti-tuberculous drugs are in urgent need so that the research on drug resistant mutations should be extended.

IDENTIFICATION GENE MUTATIONS RPOB CAUSE OF MULTIDRUG RESISTANCE TUBERCULOSIS IN HAJI ADAM MALIK HOSPITAL

Objective: The study aimed to investigate gene mutations in Mycobacterium tuberculosis causing resistance to rifampicin (RIF) in multidrug resistance TB patients in Haji Adam Malik Hospital and determined the suitability of the general pattern of mutations that occur in patients with multidrug-resistant TB (MDR-TB) in the hospital with global mutation pattern.Methods: This observational study was performed prospectively in 31 patients with MDR-TB who had data medical record results of GeneXpert MTB/ RIF positive for resistant to RIF period August–November 2016 in the hospital by taking sputum from patients. DNA extracted from sputum and then purified to be detected using polymerase chain reaction (PCR). PCR products were examined by electrophoresis.Results: The highest percentage of 81 bp rpoB gene mutation MTB is called as RIF -resistance determining region which was located at codon 516 (100%) and then was followed by mutation at codon 531 (96.77%) and codon 526 (90.32%), respectively, while the smallest percentage of mutation (12.90%) MTB at codon was 533.Conclusion: The rpoB gene shows a positive mutation-causing resistance to RIF.

Identification of rpoB, gyrA and embB Gene Mutations in Mycobacterium Tuberculosis Isolates from Retreatment Tuberculosis Patients in Nepal

Introduction: Tuberculosis remains one of the major public health problems in Nepal and increasing trend of multi drug resistant and extensively drug resistant tuberculosis (MDR /XDR TB) is a big challenge. Rapid diagnosis and appropriate treatment of MDR/XDR TB is crucial. Identification and comparison of MDR TB using rapid molecular techniques (for rpob, gyrA, rrs and embB gene mutations) with reference to drug susceptibility test (DST) were the main objectives of this study.Methodology: A cross sectional study was carried out in National TB Centre (NTC). Gene Xpert, proportion method and Line Probe Assay (LPA) were used for first and second line drugs susceptibility testing (FLD-DST and SLD-DST). A total of 29 mucopurulent sputum samples were freshly collected from retreatment TB patients (Female 41.4%, Male 58.6%) with median age of 40 years attending to the four MDR TB treatment centres of eastern and central Nepal (via private courier and directly to National TB Reference Laboratory (NRL) at NTC from April 2013 to October 2017.Results: Among 29 sputum samples (Female 41.4%; all smear+ve, Male 58.6%; 16 smear+ve and 1 smear-ve), Gene Xpert MTB/RIF assay detected 100% M. tuberculosis and rifampicin resistance (rpoB gene resistant) of which, 100% were culture positive by conventional Lowenstein–Jensen (LJ) method. FLDDST was performed on all culture positives of which, 96.6% showed MDR TB and 3.4% showed mono resistance to isonizid only. SLD-DST on 29 MDRTB strains by LPA showed 100%, 58.6%, 44.8% wild type for rrs, gyrA and emb B genes respectively. Mutation for gyrA and emb B genes was 41.4% and 51.2% respectively, rrs genes none. Twelve (Female 6, Male 6) MDR TB strains were identified as pre-XDR-TB. Chi square (χ2) for trend was used to analyze Gene Xpert, smear, FLD-DST and LPA results.Conclusion: From this study, 29(100%) MDRTB were detected from retreatment TB cases by Gene Xpert and FLDDST. Almost 41.4% MDR TB strains were detected as pre-XDR TB by LPA, which were found to be higher in 15-60 years group of females and males. Samples from retreatment TB patients need to be tested by rapid molecular techniques with reference to culture and DST.SAARC Journal of Tuberculosis, Lung Diseases and HIV/AIDS, Vol. 14, No. 2, 2017, Page: 39-50

Comprehensive identification of compensatory mutations in rifampicin-resistant Mycobacterium tuberculosis strains

Objective: To comprehensively identify compensatory mutations in rpoA, rpoB and rpoC genes of rifampicin-resistant Mycobacterium tuberculosis (RIF-r MTB) and to evaluate the effect of rifampicin-resistant mutation type and lineage background on occurrence of compensatory mutations. Methods: Published MTB whole genome sequencing data were searched and downloaded. RIF-r MTB was identified through known rifampicin-resistant mutations. Based on parallel evolutionary patterns, we identified putative compensatory mutations in the phylogenetic tree and calculated proportions of accumulating compensatory mutations in each rifampicin-resistant mutations' type and lineage background of RIF-r MTB. Statistic significance was analyzed by chi-square test. Results: A total of 8 453 global MTB whole genome sequencing data were downloaded form ENA (covering 12 countries), including 1 749 RIF-r MTB. Based on phylogenetic analysis, we totally identified 60 putative compensatory mutations (6 in rpoA gene, 16 in rpoB gene and 38 in rpoC gene), 11 of which were newly reported. RIF-R strains carrying rpoB S450L (41.7%, 279/669) had a significant higher chance to accumulate compensatory mutations than strains with other rpoB mutations (8.0%, 31/388, χ(2)=378.5, P<0.000 1). In addition, RIF-R strains from lineage 2 (34.0%, 223/656) had a significant higher chance to accumulate compensatory mutations than strains from other lineages [lineage1: 4.7%(2/43), 2/43, lineage3: 12.5%(4/32), 4/32, lineage4: 15.1%(78/517), 78/517; χ(2)=238.5, P<0.000 1]. Conclusions: Our study comprehensively identified putative rifampicin-resistant compensatory mutations of rifampicin resistance. RIF-R strains carrying rpoB S450L mutation or from lineage 2 had a significantly higher chance to accumulate compensatory mutations than strains either with other rpoB mutations or from other lineages.

Rapid detection of drug resistance in mycobacterium tuberculosis using Tag Array chip

Objective To evaluate the sensitivity and specificity of Tag Array in detecting the clinical isolates of bone and joint tuberculosis. Methods Twenty-four strains of Mycobacterium tuberculosis having sequence differences in extracted plasmids of mutant strains. The plasmid was diluted into different concentrations, and then multiplex PCR amplification was performed to analyze the sensitivity and specificity of the chip system. A total of 187 clinical isolates were collected from patients with bone and joint tuberculosis. Among them, there were 50 strains of sensitive bacteria, and 137 strains of drug-resistant. Designed, optimized and prepared chip inspection system, sequencing and phenotypic drug susceptibility results were analyzed to evaluate the sensitivity and specificity of the gene chip. Results The mutants in accordance with sequencing result in the sets with 1×103copy/μl of template concentration and above; while 29 sets showed false negative result in the sets with 1×102 copy/μl. Among the 126 rifampicin phenotype drug resistance strains, 119 strains appeared mutation in rpoB gene. In the rifampicin phenotype sensitivity strains, 9 strains of rpoB gene showed mutation, the sensitivity was 94.40%, and specificity was 86.76%. Among the 118 isoniazid phenotype drug resistance strains, 109 strains appeared mutation in relevant drug resistance gene locus, the sensitivity was 92.37%, and specificity was 81.16%. Among the 44 ethambutol phenotype drug resistance strains, 27 strains appeared mutation in locus embB306, in the ethambutol phenotype sensitivity strains, the mutation in locus embB306 was detected in 6 strains, the sensitivity was 61.36%, and specificity was 95.80%. Among the 102 levofloxacin phenotype drug resistance strains, 81 strains appeared mutation in gyrA gene, 4 of the sensitivity strains were detected mutation in gyrA gene, the sensitivity was 79.41%, and specificity was 95.29%. Among the 112 streptomycin phenotype drug resistance strains, the chip detected 101 strains appeared mutation, the sensitivity of chip detection was 90.17%, and specificity was 84.00%. Since amikacin, capreomycin and kanamycin are in cross drug resistance, among the 67 phenotype drug resistance strains, 52 strains appeared mutation in relevant drug resistance gene locus, its sensitivity was 79.10% and specificity was 90.83%. Conclusion Tag Array chip can achieve rapid, accurate and high-throughput detection of tuberculosis resistance, which has important clinical significance and feasibility. Key words: Extensively drug-resistant tuberculosis; Genetic testing; Sensitivity and specificity

In vitro antimicrobial susceptibility testing of human Brucella melitensis isolates from Ulanqab of Inner Mongolia, China

BackgroundBrucellosis is an endemic disease in the Inner Mongolia Autonomous Region of China and Ulanqab exhibits the highest prevalence of brucellosis in this region. Due to the complex nature of Brucellosis, a cure for this disease has proven to be elusive. Furthermore, the reduced susceptibility of Brucella spp. to antimicrobial agents has been reported as a potential cause of therapeutic failure. However, detailed in vitro antimicrobial susceptibility patterns pertaining to Brucella isolates from this region have not yet been published. The aim of this study was to evaluate the antibiotic susceptibility profile of Brucella melitensis clinical isolates from Ulanqab, Inner Mongolia, China.MethodsA total of 85 B. melitesis isolates were obtained from humans in Ulanqab of Inner Mongolia, China; the antimicrobial susceptibility of 85 clinical isolates to nine antibiotics was assessed using the E-test method according to the CLSI (Clinical and Laboratory Standards Institute) guidelines.ResultsAll of the tested isolates were susceptible to minocycline, sparfloxacin, doxycycline, tetracycline, ciprofloxacin, gentamicin and levofloxacin. Resistance to rifampin and cotrimoxazole was observed in 1.0% (1/85) and 7.0% (6/85) of the isolates, respectively. However, rpoB gene mutations were not observed in single isolates exhibiting resistance to rifampin.ConclusionsWe observed that B. melitensis isolates are susceptible to the majority of the tested antibiotics. Furthermore, minocycline and sparfloxacin exhibited extremely high bactericidal effects in relation to the B. melitensis isolates. The sensitivity of commonly used drugs for the treatment of brucellosis should be regularly monitored. To the best of our knowledge, this is the first report of rifampin and cotrimoxazole resistant isolates of B. melitensis in China. In summary, based on the findings from this study, we suggest that antibiotic administration and use should be rationalized to prevent future drug resistance.

Genomic Reduction at TTC Repeats in the Bacterial Genome of Treated Cases of Hansen's Disease: A Possible Survival Mechanism of Mycobacterium leprae.

Introduction:Mycobacterium leprae has a small genome and a tendency of persisting as a very low-grade infection. The authors have shown earlier, that the changes in TTC repeats, in M. leprae genome may contribute to the restriction of the pathogenicity of the bacterium and its survival strategy in case of pure neural Hansen's disease. We suspect, that a similar genomic reduction if happens in treated cases of Hansen's disease, can be a determining factor for developing persisters and relapse.Aim:The present study aimed to find out if there was any evidence of genomic reduction in treated cases of Hansen's disease that showed microbiological nonresponse.Methods:Skin biopsies were taken from treated cases of Hansen's disease at tertiary centers in Kolkata and at Raipur who had bacterial index (BI) unchanged or increased compared to their pretreatment BI. Analysis for the mutation in rpoB gene and folP1 gene were done to rule out rifampicin and dapsone resistance, respectively. The entire TTC repeat region of the bacteria was amplified by polymerase chain reaction and was subjected to sequencing. The obtained sequences were then analyzed by CLUSTALW.Results:A total of 127 patients were included in the study of which in 52 the BI remained same and 75 had an increase in BI, even after 6 months of completion of multidrug therapy. Among the samples, 2 had positive rpoB gene mutation. No mutation was found in the folP1 gene. The TTC repeat of both the rpoB-resistant samples was found to have 17 copies, which matched their pretreatment copy number. In other 125 cases, 60 cases showed no change from their pretreatment TTC number. Of those 65 samples that showed evidence of genomic reduction, 11 samples showed one copy, 41 showed 2 copies, and 13 showed 3 copies deletion. We also observed a significant regional variation.Conclusion:We concluded that there was evidence of genomic reduction, which might lead to microbiological nonresponse in treated cases of Hansen's disease. This indicated a possibility of future persistence and relapse.

Genotyping of Related Mutations to Drug Resistance in Isoniazid and Rifampin by Screening of katG, inhA and rpoB Genes in Mycobacterium tuberculosis by High Resolution Melting Method

Introduction: Tuberculosis is one of the infectious diseases and this is responsible for 3 million mortalities in the world which is increased by the rise of drug-resistant tuberculosis patients. Thus, it seems essential to detect the drug resistances in tuberculosis patients. Rifampin and isoniazid are two essential drugs for treatment of tuberculosis patients. The new High Resolution Melting method is simple, rapid and inexpensive for detection of these mutations responsible for these resistances compared to conventional methods in tuberculosis patients. Materials and methods: 2500 sputum samples were collected from patients with suspected tuberculosis referred to Iran Remedial Center over a period of 2 years in which, 1650 samples had positive smear for Mycobacterium tuberculosis. After extraction of genomic DNA from samples, High resolution melting method was used for samples based on the color of SY to-9 and PCR product were sequenced to verify the mutation. Results: Our findings showed that, 116 out of 1650 positive smear samples, were resistant to isoniazid due to mutations in katG and inhA genes, which this resistance is created by mutation of 20 samples in inhA gene and 96 samples in katG gene. Whist 65 samples had resistance to rifampin with mutation in rpoB gene. Conclusion: High resolution melting method is quick, easy and affordable without wasting time for culturing and Post PCR processes for diagnosing these resistances in tuberculosis clinical samples.