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Tullia M Simonetti - One of the best experts on this subject based on the ideXlab platform.
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evaluation of automated bactec mgit 960 system for testing susceptibility of mycobacterium tuberculosis to four major antituberculous drugs comparison with the Radiometric bactec 460tb Method and the agar plate Method of proportion
Journal of Clinical Microbiology, 2002Co-Authors: Enrico Tortoli, Marta Benedetti, Alessandra Fontanelli, Tullia M SimonettiAbstract:We evaluated the performance of BACTEC MGIT 960 for automated testing of the susceptibility of 133 strains of Mycobacterium tuberculosis to streptomycin, isoniazid, rifampin, and ethambutol. The BACTEC MGIT 960 results were compared with those obtained with the Radiometric BACTEC 460TB system, and when there was disagreement, the Method of proportion on agar plates was used as a reference Method. Strains resistant to the critical concentration of streptomycin, isoniazid, or ethambutol were also tested with a second, higher concentration. The overall agreement between the two systems was 96.7%, and the 18 discrepancies were resolved in favor of BACTEC 460TB in 11 cases and in favor of BACTEC MGIT 960 in 7, a difference which was not statistically significant. Apart from the assay's low specificity for ethambutol, which was low for the Radiometric assay as well, good sensitivity and specificity values characterized BACTEC MGIT 960. The average time required for completion of the test was 2.5 days shorter with BACTEC 460TB. In conclusion, BACTEC MGIT 960 appears to be a suitable replacement for the Radiometric Method of antimicrobial susceptibility testing of M. tuberculosis. The problem of frequent contamination of BACTEC MGIT 960 tests needs to be quickly resolved; in fact, 14 strains had to be reprocessed because of contamination.
Enrico Tortoli - One of the best experts on this subject based on the ideXlab platform.
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evaluation of automated bactec mgit 960 system for testing susceptibility of mycobacterium tuberculosis to four major antituberculous drugs comparison with the Radiometric bactec 460tb Method and the agar plate Method of proportion
Journal of Clinical Microbiology, 2002Co-Authors: Enrico Tortoli, Marta Benedetti, Alessandra Fontanelli, Tullia M SimonettiAbstract:We evaluated the performance of BACTEC MGIT 960 for automated testing of the susceptibility of 133 strains of Mycobacterium tuberculosis to streptomycin, isoniazid, rifampin, and ethambutol. The BACTEC MGIT 960 results were compared with those obtained with the Radiometric BACTEC 460TB system, and when there was disagreement, the Method of proportion on agar plates was used as a reference Method. Strains resistant to the critical concentration of streptomycin, isoniazid, or ethambutol were also tested with a second, higher concentration. The overall agreement between the two systems was 96.7%, and the 18 discrepancies were resolved in favor of BACTEC 460TB in 11 cases and in favor of BACTEC MGIT 960 in 7, a difference which was not statistically significant. Apart from the assay's low specificity for ethambutol, which was low for the Radiometric assay as well, good sensitivity and specificity values characterized BACTEC MGIT 960. The average time required for completion of the test was 2.5 days shorter with BACTEC 460TB. In conclusion, BACTEC MGIT 960 appears to be a suitable replacement for the Radiometric Method of antimicrobial susceptibility testing of M. tuberculosis. The problem of frequent contamination of BACTEC MGIT 960 tests needs to be quickly resolved; in fact, 14 strains had to be reprocessed because of contamination.
R Martin - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the bactec mgit 960 and the mb bact systems for recovery of mycobacteria from clinical specimens and for species identification by dna accuprobe
Journal of Clinical Microbiology, 2000Co-Authors: Fernando Alcaide, M A Benitez, Josep M Escriba, R MartinAbstract:A total of 120 mycobacterial isolates were recovered from 1,068 clinical specimens. Of these, 82.5% were in MGIT 960, 83.3% were in MB/BacT, 80% were in BACTEC 460, and 70% were on Lowenstein-Jensen medium. Mean times to detection of Mycobacterium tuberculosis (n = 96) were significantly shorter with MGIT 960 (12.6 days, P = 0.003) and BACTEC 460 (11.8 days, P < 0.001) than with MB/BacT (15.9 days). Although, MGIT 960 showed the lowest rate of recovery of M. kansasii genotype I (64.3%), the earliest growth was detected with this system (8.9 days). Low and similar rates of contamination were obtained with MGIT 960 (3.3%) and MB/BacT (3%). The AccuProbe test for identification showed excellent sensitivities with MGIT 960 (96.8%) and MB/BacT (100%) cultures. In addition to being nonRadiometric, both MGIT 960 and MB/BacT are accurate, rapid, and labor-saving detection systems which could replace the Radiometric Method.
Sin Yew Wong - One of the best experts on this subject based on the ideXlab platform.
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novel mutations within the embb gene in ethambutol susceptible clinical isolates of mycobacterium tuberculosis
Antimicrobial Agents and Chemotherapy, 2004Co-Authors: Ann S G Lee, Siti Noor Khadijah Othman, Sin Yew WongAbstract:Ethambutol (EMB) [(S,S′)-2,2′-(ethylenediimino)di-1-butanol] is a first-line drug used for antituberculosis therapy. It is often used in combination with isoniazid, rifampin, pyrazinamide, and streptomycin. Membrane-associated arabinosyl transferases have been implicated as the targets for EMB (2, 3, 14, 15). The Mycobacterium tuberculosis emb operon is a gene cluster of three contiguous genes, namely, embC, embA, and embB, which encode mycobacterial arabinosyl transferases (26). These enzymes are involved in the polymerization of the cell wall arabinan (4, 6, 9, 24, 25, 32). Inhibition of arabinan synthesis by EMB results in the accumulation of mycolic acids, leading to cell death. Alterations at codon 306 of embB have been identified as being the most common alteration in EMB-resistant M. tuberculosis clinical isolates (8, 12, 17-20, 23, 29). Initial work on 51 EMB-resistant isolates had shown that 89% of these isolates had alterations at residue 306 of embB, but these alterations were not detected in 30 EMB-susceptible isolates (23). A subsequent study confirmed this high frequency of embB306 alterations, with 67% of 75 EMB-resistant isolates having mutations not found in EMB-susceptible strains (19). This led to several groups developing targeted strategies for the detection of embB306 alterations (7, 16, 21, 30). Amino acids within the EMB resistance-determining region of EmbB proteins are well conserved among mycobacterial species, including those from M. tuberculosis, M. leprae, and M. smegmatis (2), and mutations within this region have been detected in EMB-resistant isolates of M. tuberculosis. The aim of this present work was to screen all regions of the embB gene with previously reported mutations in order to assess the contribution of mutations within this gene to EMB resistance in M. tuberculosis clinical isolates from Singapore. Drug susceptibility testing was done using the BACTEC 460 Radiometric Method (Becton Dickinson, Towson, Md.) (2.5 μg/ml). Twenty-five consecutive M. tuberculosis isolates resistant to EMB and 20 EMB-susceptible isolates from Singapore were collected as previously described (5, 10). DNA extracted from the isolates was analyzed by amplifying four fragments, using the PCR primers shown in Table Table1.1. The PCR products were purified (QIAquick PCR purification kit or QIAquick gel extraction kit; QIAGEN) and directly sequenced using the BigDye Terminator sequencing kit and the ABI PRISM 377 automated sequencer (PE Biosystems, Branchburg, N.J.). Confirmation of mutations was done by reamplification and resequencing. TABLE 1. Oligonucleotide primer sequences for amplification of the embB genea IS6110 profiling was done according to standard procedures to determine if the isolates were epidemiologically independent (28). All isolates with the same nucleotide substitutions in this study were deemed to be epidemiologically unassociated as they had distinct IS6110 fingerprints. Overall, mutations in the embB gene were detected in 17 (68%) of the 25 EMB-resistant isolates (Table (Table2).2). Mutations at embB306 were detected in 12 of these 25 (48%) EMB-resistant isolates. Notably, all of the 12 EMB-resistant isolates with embB306 mutations were also resistant to isoniazid. All five EMB-resistant isolates with mutations at codon 497 were resistant to at least three antituberculosis drugs. Three isolates monoresistant to EMB had no detectable mutations in embB. TABLE 2. Mutations in the embB gene in clinical isolates of M. tuberculosis This is the first report of a double substitution (ATG→ATM, where M represents the nucleotides A and C), resulting in a Met→Ile alteration at the frequently altered codon 306 of embB in an EMB-susceptible isolate (Table (Table2).2). This isolate was resistant to both isoniazid and rifampin. In addition, three other alterations were also detected in EMB-susceptible isolates, G406D and two novel mutations, M423I and A659T. The isolate with the G406N substitution was also resistant to isoniazid and rifampin, while the isolate with the M423I alteration was monoresistant to isoniazid and the isolate with the A659T alteration was monoresistant to streptomycin. In total, alterations in the embB gene were detected in 4 (20%) of the 20 EMB-susceptible isolates (Table (Table22). There is a possibility that these mutations may have occurred in susceptible isolates due to cross-contamination of the PCR product, heteroresistance involving mixed cultures, or errors in the susceptibility testing, though every effort was undertaken to avoid this. Alterations in embB in EMB-susceptible isolates at codons other than codon 306 have been documented in only two isolates with the G406D alteration (20) and one isolate with the S347T alteration (8). This paucity of information is due in part to some studies targeting only embB306 (17, 29), several reporting no mutations (1, 12, 19, 23), and others not including EMB-susceptible isolates (22, 31). Interestingly, all three EMB monoresistant isolates in this present study did not have any detectable alterations in embB. In contrast, all 58 EMB-susceptible isolates in this and other studies with embB alterations were resistant to other antituberculosis drugs as well (8, 17, 20, 29). These observations support the hypothesis that a target other than EmbB may exist for EMB which may be activated during combination treatment with other first-line antituberculosis drugs, resulting in susceptibility to EMB (17). Importantly, if all alterations of embB306 are considered polymorphisms, then only a minority of EMB-resistant isolates would be mutated. A similar scenario was observed in studies defining the role of the katG gene in isoniazid resistance in M. tuberculosis. Members of our group and others have shown that the predominant alteration in katG is R463L, which is detected in both isoniazid-resistant and -susceptible isolates and hence is considered a polymorphism and an unreliable indicator of isoniazid resistance (11, 13, 27). Thus, it is imperative for all studies elucidating the molecular mechanisms of drug resistance in M. tuberculosis to include drug-susceptible isolates as controls. Another interesting finding of this study was the presence of resistance to other antituberculosis drugs when alterations of embB were present. Further investigations are necessary in order to understand the involvement of these drugs in the molecular mechanism of EMB resistance. In conclusion, alterations at embB306 may not confer resistance to EMB but may be common polymorphisms in clinical isolates of M. tuberculosis. The clinical significance of this alteration is dubious, and further evaluation of EMB-susceptible isolates from other geographic regions is warranted.
Marta Benedetti - One of the best experts on this subject based on the ideXlab platform.
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evaluation of automated bactec mgit 960 system for testing susceptibility of mycobacterium tuberculosis to four major antituberculous drugs comparison with the Radiometric bactec 460tb Method and the agar plate Method of proportion
Journal of Clinical Microbiology, 2002Co-Authors: Enrico Tortoli, Marta Benedetti, Alessandra Fontanelli, Tullia M SimonettiAbstract:We evaluated the performance of BACTEC MGIT 960 for automated testing of the susceptibility of 133 strains of Mycobacterium tuberculosis to streptomycin, isoniazid, rifampin, and ethambutol. The BACTEC MGIT 960 results were compared with those obtained with the Radiometric BACTEC 460TB system, and when there was disagreement, the Method of proportion on agar plates was used as a reference Method. Strains resistant to the critical concentration of streptomycin, isoniazid, or ethambutol were also tested with a second, higher concentration. The overall agreement between the two systems was 96.7%, and the 18 discrepancies were resolved in favor of BACTEC 460TB in 11 cases and in favor of BACTEC MGIT 960 in 7, a difference which was not statistically significant. Apart from the assay's low specificity for ethambutol, which was low for the Radiometric assay as well, good sensitivity and specificity values characterized BACTEC MGIT 960. The average time required for completion of the test was 2.5 days shorter with BACTEC 460TB. In conclusion, BACTEC MGIT 960 appears to be a suitable replacement for the Radiometric Method of antimicrobial susceptibility testing of M. tuberculosis. The problem of frequent contamination of BACTEC MGIT 960 tests needs to be quickly resolved; in fact, 14 strains had to be reprocessed because of contamination.
