Significance of Mutations in embB Codon 306 for Prediction of Ethambutol Resistance in Clinical Mycobacterium tuberculosis Isolates

Abstract

We analyzed 159 Mycobacterium tuberculosis isolates (101 ethambutol [EMB]-resistant strains, 33 multidrugresistant but not EMB-resistant strains, and 25 fully susceptible strains) for the presence of mutations in embB codon 306 (embB306). Mutations were detected only in EMB-resistant strains (n ‫؍‬ 69; 68%), thus confirming the significance of embB306 mutations for the prediction of resistance to EMB.Drug-resistant tuberculosis (TB) has become a major public health problem in several regions around the world (14). The third report of the WHO/IUATLD Global Project on AntiTuberculosis Drug Resistance confirmed the serious magnitude and widespread occurrence of drug resistance. Documented rates of drug-resistant TB have reached tremendous levels of up to 57% among new cases (14).The rapid determination of drug resistance is the prerequisite for the initiation of effective chemotherapy to ensure successful treatment of the patient and to prevent the further spread of drug-resistant isolates (7). Based on the knowledge that the development of drug resistance in Mycobacterium tuberculosis complex isolates is the result of random genetic mutations in particular genes conferring resistance (15), molecular assays which allow the prediction of drug resistance in clinical isolates within 1 working day have been established. Thus, these assays are potentially the most rapid methods for the detection of drug resistance (6).In the case of ethambutol (EMB), which, in combination with isoniazid, rifampin, and pyrazinamide, is a key component of the first-line anti-TB treatment regimen, resistance was most frequently associated with mutations in the embCAB operon and particularly with mutations in embB codon 306 (embB306) (15). Overall, approximately 60% of EMB-resistant M. tuberculosis isolates carry a mutation in embB306 (15). The determination of alterations of this codon was suggested as a rapid screening method for the detection of EMB resistance in clinical isolates (3,8,13).More recently, however, discrepancies between the results of genotypic and phenotypic EMB resistance testing have raised concerns about the accuracy of molecular assays based on the detection of point mutations in embB306 for the prediction of EMB resistance (2, 4, 5, 11). Discordant results were especially reported for multidrug-resistant (MDR) strains phenotypically susceptible to EMB (5), and in a recent paper, Hazbón and colleagues described for embB codon 306 mutations "a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance" (2).To further investigate this question, we analyzed a large collection of 159 M. tuberculosis strains isolated in Germany in the year 2001 for the presence of mutations in embB306 and the association with phenotypic resistance to EMB. Resistance to the key antimycobacterial drugs was determined at the Supranational Reference Laboratory (SRL) in Borstel, Germany, by using the proportion method on Löwenstein-Jensen medium (critical concentration, 2.0 g/ml for EMB) (1) and/or the ...

Tuberculosis ethambutol resistance: Concordance between phenotypic and genotypic test results

“…Statistical analysis revealed a significant association between EMB resistance and embB306 mutations in univariate analysis (p < 0.0001), with embB306 mutations the only significant factor in multivariate analysis (p < 0.0001). Thus, our study confirms previous investigations that describe a clear correlation between mutations in emb306 and EMB resistance, 11,21 without any indication of an influence on the resistance to other antituberculous drugs as suggested by other authors. 13,15,16 This high concordance between both assays was, however, determined only after repeating conventional drug susceptibility testing, pointing to problems with conventional diagnostics of EMB resistance.…”

“…9 However, if clinical isolates are considered, the percentage of EMB resistant strains which show mutations in embB306 ranges from 30 to 68%. 10,11 Additionally, a number of recent investigations described the occurrence of embB306 mutations in EMB susceptible strains, [12][13][14] and several studies reporting an association with broad drug resistance rather than with EMB resistance alone. 15,16 These contradictory results cast doubt on the potential use of embB306 mutations as molecular markers for predicting EMB resistance in clinical isolates.…”

Performance Assessment of The Polymerase Chain Reaction-restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of XDR Tuberculosis in Clinical Isolates of Mycobacterium Tuberculosis

“…Shen et al (6), Plinke et al (3) and numerous authors have exclusively evaluated mutations of this gene in the clinical strains by examining codon embB306. Therefore, study of codon embB497 is neglected with the aim of reducing further simplification of the procedure, and making it possible to perform routinely.…”

“…Thus, culture results do not comply fully with the molecular results (determine the occurrence of mutations with low sensitivity, besides high specificity). The precision of determining resistant strains by molecular method has been always debated in previous research (3)(4)(5)(6)11). In most of the studies conducted by other researchers, no mutation in sensitive strains was found (10).…”

“…Mycobacterium mb CAB operon contains three joined genes of embA, embB and embC that encode three arabinosyl transferases homologous. The embB gene encodes arabinosyl transferase, which is the target protein of EMB (3)(4)(5). Ethambutol resistance is thought to occur due to mutations in embB gene of Mycobacterium tuberculosis.…”