Detection of embB codon 306 mutations in ethambutol resistant Mycobacterium tuberculosis directly from sputum samples: a low-cost, rapid approach

Rinder, Heinz; Mieskes, Klaus T.; Tortoli, Enrico; Richter, Elvira; Casal, Manuel; Vaquero, Manuel; Cambau, Emmanuelle; Feldmann, Knut; Löscher, Thomas

doi:10.1006/mcpr.2000.0339

Cited by 25 publications

(19 citation statements)

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“…The association between embB306 mutations and EMB resistance in clinical M. tuberculosis isolates is so strong that it has been proposed as a marker for EMB resistance in diagnostic tests (13,33,36,43,52,54). Thus, it was a surprise when Mokrousov et al (37) first described 48 clinical M. tuberculosis isolates from Russia that were susceptible to EMB yet that had mutations in embB306.…”

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confidence: 99%

Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS 6110 Clustering Rather than Ethambutol Resistance

Hazbón

Bobadilla-del-Valle

Guerrero

et al. 2005

Antimicrob Agents Chemother

103

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Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P ‫؍‬ 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P ‫؍‬ 0.008) and katG315 (odds ratio, 1.99; P ‫؍‬ 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.The antibiotic ethambutol (EMB) appears to inhibit the growth of both Mycobacterium tuberculosis and Mycobacterium smegmatis by blocking the synthesis of arabinogalactan. Arabinogalactan biosynthesis is dependent on the activity of the embABC gene cluster, which encodes the arabinotransferases that mediate the polymerization of arabinose into arabinan. Several lines of evidence suggest that EMB exerts its toxic effect on mycobacteria by inhibiting the embABC-encoded proteins (26,27,34,35,46), and mutations in embABC also appear to play a key role in the development of EMB resistance in both M. tuberculosis and M. smegmatis (34). Associations between EMB resistance and mutations in embA, embB, and embC have been reported in clinical strains of M. tuberculosis (41), and mutations in codon 306 of the embB gene (embB306) in M. tuberculosis are seen in approximately 50% of all EMB-* Corresponding author. Mailing address:

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Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS 6110 Clustering Rather than Ethambutol Resistance

Hazbón

Bobadilla-del-Valle

Guerrero

et al. 2005

Antimicrob Agents Chemother

103

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“…For isoniazid resistance, the sensitivity of the method is only 53.8%. As with other genotype-based resistance prediction tests, a drawback in characterizing katG codon 315 mutations and inhA mutations, as well as emb mutations for ethambutol resistance, is that no prediction can be made for samples containing the nonmutated genotypes (19). Therefore, the diagnostic categories cannot be "resistant" and "sensitive" but must be "resistant" and "no prediction possible."…”

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confidence: 99%

“…Nucleic acid amplification-based techniques are potentially the most rapid and sensitive methods for detection, identification, and susceptibility testing and are theoretically able to provide a sameday diagnosis from clinical samples (10,15,17,19). These methods can potentially reduce the diagnostic time from weeks to days (20).…”

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Direct Detection of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis in Auramine-Rhodamine-Positive Sputum Specimens by Real-Time PCR

et al. 2004

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Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (>0.1 g/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auraminerhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens.

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“…EMB seems to exert its toxic effect by inhibiting the embABC-encoded proteins, and mutations in the embB gene appear to play a major role in the development of EMB resistance in Mycobacterium tuberculosis (13). The marked clinical association between embB codon 306 mutations and EMB resistance in M. tuberculosis at one time led to its proposal as a marker for EMB resistance in diagnostic tests (6,8,11,15). However, discrepancies between the results of genotypic and phenotypic EMB resistance testing have raised questions about the accuracy of molecular assays based on the detection of point mutations in embB codon 306 for prediction of EMB resistance (3,4,7,14).…”

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Lack of Correlation betweenembBMutation and Ethambutol MIC inMycobacterium tuberculosisClinical Isolates from China

Shi

Zhang

Otomo

et al. 2007

Antimicrob Agents Chemother

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Seventy-four Mycobacterium tuberculosis clinical isolates from China were subjected to drug susceptibility testing using ethambutol, isoniazid, rifampin, and ofloxacin. The results revealed that the presence of embB mutations did not correlate with ethambutol resistance but was associated with multiple-drug resistance, especially resistance to both ethambutol and rifampin.Ethambutol (EMB), often used in combination with isoniazid, rifampin, and pyrazinamide, is a key drug of first-line antituberculosis treatment. EMB seems to exert its toxic effect by inhibiting the embABC-encoded proteins, and mutations in the embB gene appear to play a major role in the development of EMB resistance in Mycobacterium tuberculosis (13). The marked clinical association between embB codon 306 mutations and EMB resistance in M. tuberculosis at one time led to its proposal as a marker for EMB resistance in diagnostic tests (6,8,11,15). However, discrepancies between the results of genotypic and phenotypic EMB resistance testing have raised questions about the accuracy of molecular assays based on the detection of point mutations in embB codon 306 for prediction of EMB resistance (3,4,7,14). Hazbon et al. (3) has reported that for embB codon 306 mutations, there is "a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance."For DNA sequencing of the embB gene (primer set includes forward, 5Ј-CGGCATGCGCCGGCTGATTC, and reverse, 5Ј-TCCACAGACTGGCGTCGCTG) from 141 EMB-resistant and 40 EMB-sensitive clinical isolates from Henan Province, China, ABI Prism Big Dye terminator sequencing kits were used. We found that 45.2% of EMB-resistant isolates harbored embB codon 306 mutations (ATG to ATA, GTG, ATT, CTG, or ATC [five types]) (12). We also found that 15% (6/40) of EMB-susceptible isolates had embB gene mutations (the breakpoint concentration of EMB is 2 g/ml in L-J medium) in 2000. After a previous analysis by denaturing highpressure liquid chromatography (DHPLC) of drug resistance genes in M. tuberculosis in our laboratory (9, 10), we went back to test these isolates and found that the DHPLC results for the embB gene were completely consistent with those of DNA sequencing. Figure 1 shows the DHPLC and DNA sequencing results for the six isolates that were EMB sensitive but harbored embB mutations. Results similar to those we obtained for streptomycin resistance were obtained (R. Shi, J. Zhang, C. Li, Y. Kazumi, and I. Sugawara, unpublished data). We also found that 22% (16/72 EMB-sensitive clinical isolates from an affiliated hospital of the Beijing Tuberculosis and Lung Tumor Research Institute) possessed embB mutations in 2006. We tested the EMB MICs for these 16 isolates, and all were less than 2 g/ml, but the MIC for four isolates was 0.125 g/ml (data not shown).

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Detection of embB codon 306 mutations in ethambutol resistant Mycobacterium tuberculosis directly from sputum samples: a low-cost, rapid approach

Cited by 25 publications

References 10 publications

Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS 6110 Clustering Rather than Ethambutol Resistance

Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS 6110 Clustering Rather than Ethambutol Resistance

Direct Detection of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis in Auramine-Rhodamine-Positive Sputum Specimens by Real-Time PCR

Lack of Correlation betweenembBMutation and Ethambutol MIC inMycobacterium tuberculosisClinical Isolates from China

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