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1.
A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates 总被引:3,自引:0,他引:3
Rindi L Bianchi L Tortoli E Lari N Bonanni D Garzelli C 《Journal of microbiological methods》2003,55(3):797-800
A real-time PCR genotypic assay was developed for the detection of isoniazid (INH) resistance in Mycobacterium tuberculosis. The assay detects mutations C(-15)T and, possibly, G(-24)T in the regulatory region of the inhA gene and proved as sensitive and specific as nucleotide sequencing in all the clinical isolates tested. Our assays mapped the mutations efficiently in 10 out of 35 resistant isolates, thereby covering 29% of all resistant strains. 相似文献
2.
Multidrug-resistant (MDR) strains of Mycobacterium tuberculosis have emerged worldwide. In many countries and regions, these resistant strains constitute a serious threat to the efficacy of tuberculosis control programs. An important element in gaining control of this epidemic is developing an understanding of the molecular basis of resistance to the most important antituberculosis drugs: isoniazid, rifampin, and pyrazinamide. On the basis of this information, more exacting laboratory testing, and ultimately more appropriate and timely treatment regimens, can be developed. 相似文献
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Hiroki Ando Tohru Miyoshi‐Akiyama Shinya Watanabe Teruo Kirikae 《Molecular microbiology》2014,91(3):538-547
Drug resistance in Mycobacterium tuberculosis (Mtb) is caused by mutations in restricted regions of the genome. Mutations in katG, the promoter region of the mabA–inhA operon, and inhA are those most frequently responsible for isoniazid (INH) resistance. Several INH‐resistant (INHr) Mtb clinical isolates without mutations in these regions have been described, however, indicating that there are as yet undetermined mechanisms of INH resistance. We identified the mabAg609a silent mutation in a significant number of INHr Mtb clinical isolates without known INH resistance mutations. A laboratory strain, H37Rv, constructed with mabAg609a, was resistant to INH. We show here that the mabAg609a mutation resulted in the upregulation of inhA, a gene encoding a target for INH, converting the region adjacent to the mutation into an alternative promoter for inhA. The mabAg609a silent mutation results in a novel mechanism of INH resistance, filling in a missing piece of INH resistance in Mtb. 相似文献
7.
Anti-Mycobacterium tuberculosis drug-resistance, mainly multi-drug resistance (MDR-TB), represents an important public health problem in several countries. Aim of our study is to identify the presence of these mutations in M. tuberculosis isoniazid- and rifampin-resistant strains isolated in our Institute; to evaluate linkage between type of mutation and level of resistance; to determine the usefulness of easy molecular techniques for rapid detection of such mutations on body specimens. Isoniazid- and rifampin-resistance was tested on 67 M. tuberculosis strains by Single-Strand Conformation Polymorphism (SSCP) and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assays, using HaeIII, PstuI, BsteII, BstuI enzymes. Drug-resistance of control strains was determined by cultural techniques (fluorimetry- BACTEC 9120). Cultural assay showed isoniazid- and rifampin-resistance in 6.12 and 2%, respectively (data confirmed by SSCP assay). Mutation of katG, linked to isoniazid resistance, was detected using BstuI enzyme, and mutation of rpoB, expression of reduced sensitivity to rifampin, using HaeIII. 15 body specimens, M. tuberculosis-positive to conventional assays, were tested by SSCP technique. Epidemiologic reports of numerous cases of tuberculosis due to MDR strains induce to detect quickly both Mycobacteria and drug-resistance, in order to start prompt effective therapy. On this basis, molecular assays are useful for a rapid therapeutic decision. 相似文献
8.
抗结核一线药物异烟肼是应用最广泛的抗结核药物之一,自1952年应用于临床以来,异烟肼就成了治疗结核和潜在感染的基础药物.有报道,我国异烟肼耐药已排在首位.结核分枝杆菌对异烟肼耐药的分子机制十分复杂,涉及katG、inhA、kasA、ndh、axyR等多种基因,本研究仅就此方面的研究作一综述. 相似文献
9.
We present a robust and simple method for the direct detection of multiple point mutations in the Mycobacterium tuberculosis rpoB gene during the development of rifampin (RIF) resistance using an electrochemical genosensor. The device contained five different capture probes which are designed to hybridize with several sequence segments within the bacterial rpoB gene hotspot region. Point mutations were detected by monitoring the guanine oxidation with differential pulse voltammetry after hybridization between PCR amplicons and inosine modified capture probes at graphite surface. Changes in the peak voltage corresponding to guanine oxidation provide an electrochemical signal for hybridization that can be used to determine the presence of point mutations conferring rifampin resistance. The analytical parameters (sensitivity, selectivity and reproducibility) were evaluated. High selective discrimination against point mutation of bacteria at hot-spot region was observed. Several mutations were detected at several parts of the amplicon from 21 positive samples. 相似文献
10.
Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis 总被引:2,自引:0,他引:2
Martin A Montoro E Lemus D Simboli N Morcillo N Velasco M Chauca J Barrera L Ritacco V Portaels F Palomino JC 《Journal of microbiological methods》2005,63(2):145-150
The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on L?wenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities. 相似文献
11.
Many of the deaths caused by tuberculosis (TB) in the world are due to wrong or late diagnosis. The World Health Organization (WHO) calls for better and cheaper TB tests method for this reason. In this paper, a new multi-channel series piezoelectric quartz crystal (MSPQC) sensor system was developed for rapid growth and detection of Mycobacterium tuberculosis. It is an automatic continuous monitoring system. The system was used to detect TB based on the volatile metabolic products NH(3) and CO(2) during the growth of M. tuberculosis. The metabolic products, diffusing from the medium into the KOH absorbing solution, resulted in the conductance change of the absorbing solution detected by the MSPQC sensitively. The frequency shift versus time response curves were recorded by self-developed software. Frequency detection time (FDT) corresponding to -100Hz in frequency shift value was used as a parameter to quantitatively determine M. tuberculosis H37Ra (an avirulent strain). H37Ra and 40 strains clinic positive samples were detected by the proposed system successfully. As for H37Ra, the FDT had a linear relationship with the logarithm of its initial concentration in the range of 10(2)-10(7) colony forming units (cfu)ml(-1) (R=-0.998) and the detection limit was low to 10cfuml(-1). 4% NaOH solution that can kill contaminating microorganisms and make M. tuberculosis alive was used as pretreatment reagent to provide selectivity to this method. Comparative tests were also carried out by using BACTECtrade mark MGITtrade mark 960 and conventional Lowenstein-Jensen (L-J) slants. The results showed that the proposed system was quicker than BACTECtrade mark MGITtrade mark 960 and it is also cheaper and will be widely used in TB tests in the world. 相似文献
12.
The alarmingly worsening epidemics of drug-resistant tuberculosis (TB) call urgent need for a simple method for the rapid
detection of drug-resistant TB in clinical settings. In an attempt to establish a rapid procedure for laboratory diagnosis
of TB and investigate the local TB epidemiology, molecular line probe assay of the Genotype MTBDRplus was used to identify
Mycobacterium tuberculosis complex (MTBC) and detect mutations conferring resistance to two most active first-line drugs against
TB: Rifampin and Isoniazid. 96 acid-fast bacillus (AFB) smear- positive sputums and 18 PCR-positive non-sputum specimens have
been determined for the MTBC and resistance to Rifampin and Isoniazid. The MTBC detection rates in two sources of specimens
were 93.8% (90/96) and 77.8% (14/18) respectively. The overall drug resistance (Rifampin or Isoniazid) occurred in 34.6% (36/104).
Resistance to rifampin (RMP) was 28.8% (30/104) and 25% (26/104) was to Isoniazid (INH), in which high level drug resistance
accounted for 88.5% (23/26) and low level drug resistance accounted for 7.7% (2/26). Multidrug resistance (MDR), defined as
resistant to both RMP and INH, was found in 19.2% (20/104) of clinical samples, which was double that of official statistics.
In addition, 63.3% (19/30) RMP-resistant mutations were identified in the region of RopB 530–533 and 57.9% (11/19) were the
S531L mutation. 84.6% (22/26) of resistance to INH was mediated by Kat S315T1 mutations which conferred the high-level resistance
to INH. The Genotype MTBDRplus line probe assay is a suitable and applicable method for establishing the rapidness in detection
of drug-resistant TB in clinical laboratory. It will be a valuable addition to the conventional TB diagnostic approaches. 相似文献
13.
Michelon CT Rosso F Schmid KB Sperhacke RD Oliveira MM Kritski AL Rezende Jr L Costa ER Ribeiro AW Verza M Cafrune PI Silva MS Kuhleis D Zaha A Rossetti ML 《Memórias do Instituto Oswaldo Cruz》2011,106(2):194-199
Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis. 相似文献
14.
In order to characterize mutations causing rifampicin and isoniazid resistance of M. tuberculosis in Syria, 69 rifampicin resistant (Rif(r)) and 72 isoniazid resistant (Inh(r)) isolates were screened for point mutations in hot spots of the rpoB, katG and inhA genes by DNA sequencing and real time PCR. Of 69 Rif(r) isolates, 62 (90%) had mutations in the rifampin resistance determining region (RRDR) of the rpoB gene, with codons 531 (61%), 526 (13%), and 516 (8.7%) being the most commonly mutated. We found two new mutations (Asp516Thr and Ser531Gly) described for the first time in the rpoB-RRDR in association with rifampicin resistance. Only one mutation (Ile572Phe) was found outside the rpoB-RRDR. Of 72 Inh(r) strains, 30 (41.6%) had a mutation in katGcodon315 (with Ser315Thr being the predominant alteration), and 23 (32%) harbored the inhA(-15C-->T) mutation. While the general pattern of rpoB-RRDR and katG mutations reflected those found worldwide, the prevalence of the inhA(-15C-->T mutation was above the value found in most other countries, emphasizing the great importance of testing the inhA(-15C-->T) mutation for prediction of isoniazid resistance in Syria. Sensitivity of a rapid test using real time PCR and 3'-Minor groove binder (MGB) probes in detecting Rif(r) and Inh(r) isolates was 90% and 69.4%, respectively. This demonstrates that a small set of MGB-probes can be used in real time PCR in order to detect most mutations causing resistance to rifampicin and isoniazid. 相似文献
15.
Ando H Kitao T Miyoshi-Akiyama T Kato S Mori T Kirikae T 《Molecular microbiology》2011,79(6):1615-1628
Isoniazid (INH) is a key agent in the treatment of tuberculosis. In Mycobacterium tuberculosis, INH is converted to its active form by KatG, a catalase-peroxidase, and attacks InhA, which is essential for the synthesis of mycolic acids. We sequenced furA-katG and fabG1-inhA in 108 INH-resistant (INH(r) ) and 51 INH-susceptible (INH(s) ) isolates, and found three mutations in the furA-katG intergenic region (Int(g-7a) , Int(a-10c) and Int(g-12a) ) in four of 108 INH(r) isolates (4%), and the furA(c41t) mutation with an amino acid substitution in 18 INH(r) isolates (17%). These mutations were not found in any of 51 INH(s) isolates tested. We reconstructed these mutations in isogenic strains to determine whether they conferred INH resistance. We found that the Int(g-7a) , Int(a-10c) and Int(g-12a) single mutations in the furA-katG intergenic region decreased katG expression and conferred INH resistance. In contrast, the furA(c41t) mutation was not sufficient to confer INH resistance. These results suggested that downregulation of katG is a mechanism of INH resistance in M. tuberculosis and that mutations in the furA-katG intergenic region play a role in this resistance mechanism. 相似文献
16.
Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTECTM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTECTM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTECTM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube. 相似文献
17.
Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates 总被引:7,自引:0,他引:7
Mokrousov I Bhanu NV Suffys PN Kadival GV Yap SF Cho SN Jordaan AM Narvskaya O Singh UB Gomes HM Lee H Kulkarni SP Lim KC Khan BK van Soolingen D Victor TC Schouls LM 《Journal of microbiological methods》2004,57(3):323-335
A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds. 相似文献
18.
Yongzhong Wang Xiaoqin Liu Dongmei Rui Minzhi Zhu Hongyu Zhang Chunhua Chen Hongxia Zhao Zhen Zhu Xin Xu Liming Zheng 《Acta biochimica et biophysica Sinica》2014,(1):78-81
Mycobacterium tuberculosis (MTB) infection remains a serious infectious disease worldwide, causing 8.8 million new infections and 1.45 million deaths in 2010 [1]. The emergence of drug-resistant strains of MTB poses a significant threat to the control of the disease globally. Multidrugresistant MTB (MDR-TB), defined as being resistant to at least rifampicin (RMP) and isoniazid (1NH), 相似文献
19.
Ali Nour-Neamatollahi Seyed Davar Siadat Shamsi Yari Alireza Hadizadeh Tasbiti Nayereh Ebrahimzadeh Farzam Vaziri Abolfazl Fateh Morteza Ghazanfari Farid Abdolrahimi Shahin Pourazar Ahmadreza Bahrmand 《Saudi Journal of Biological Sciences》2018,25(3):418-425
Mycobacterium tuberculosis, acid fast bacilli from the family of Mycobacteriaceae, is the causative agent of most cases of tuberculosis. Tuberculosis, as a communicable disease, remains a serious public health threat, killing more than one million people globally every year. Primary diagnosis of tuberculosis bacilli (TB) relies mainly on microscopic detection of acid fast bacilli (AFB), but the method suffers from low sensitivity and the results largely depend on the technician’s skill. New diagnostic tools are necessary to be introduced for rapid and accurate detection of the bacilli in sputum samples. We, in collaboration with Anda Biologicals, have developed a new platform, named as “Patho-tb”, for rapid detection of AFB with high sensitivity and with low dependence on human skills. Evaluation of Patho-tb test performance was done in two settings: (1) primary field study conducted using 38 sputa from high TB prevalence area of Iran (Zabol city near to the Afghanistan border), and (2) main study conducted using 476 sputa from Tehran, capital of Iran. Patho-tb was applied for processed sputum samples in parallel with routine diagnostic methods (including AFB microscopy, culture and PCR). All test results were compared to final clinical diagnostic state of an individual and diagnostic sensitivity (DSe), specificity, positive predictive value, negative predictive value and accuracy of each test results were calculated using standard formulations. Analytical sensitivity and specificity of the Patho-tb test were also determined. Calculated values for five above mentioned parameters are as follows: for field study: AFB (DSe: 29.6, DSp: 81.8, PPV: 80, NPV: 23.1, AC: 44.7), Patho-tb (DSe: 63, DSp: 72.7, PPV: 85, NPV: 44.4, AC: 65.8), and for main study: AFB (DSe: 86.1, DSp: 99.4, PPV: 98.5, NPV: 93.9, AC: 95.2), Patho-tb (DSe: 97.4, DSp: 92.9, PPV: 86.5, NPV: 98.7, AC: 94.3). Reproducibility of Patho-tb test results were near to 100% (Cohen’s kappa value between 0.85 and 1). The detection limit of Patho-tb test with 100% positivity rate was 3 × 103 cells/ml of sputum. In the field study, Patho-tb test was 33.4% more sensitive than AFB microscopy, while the improvement was only 11.3% during the main study. Patho-tb results are easy to interpret and the test can be merged with other screening tests, like AFB. Totally, Patho-tb test alone or in conjunction with AFB microscopy is a useful screening tool for TB detection especially in poor geographical lab conditions. 相似文献