Multicenter Evaluation of Ethambutol Susceptibility Testing of Mycobacterium tuberculosis by Agar Proportion and Radiometric Methods

Abstract

Reproducibility of ethambutol (EMB) susceptibility test results forMycobacterium tuberculosis has always been difficult for a variety of reasons, including the narrow range between the critical breakpoint for EMB resistance and the MIC for susceptible strains, borderline results obtained with the BACTEC 460TB method, the presence of microcolonies determined using the agar proportion (AP) method, and a lack of agreement between these two testing methods. To assess the frequency of these problems, M. tuberculosis drug susceptibility data were collected in a multicenter study involving four laboratories. Resistant, borderline, and susceptible isolates were shared among the laboratories to measure interlaboratory test agreement. Half of isolates determined by BACTEC 460TB to be resistant were determined to be susceptible by the AP method. Isolates determined to be resistant to EMB by both BACTEC 460TB and AP methods were almost always resistant to isoniazid. Results from isolates tested by the BACTEC 460TB method with an EMB concentration of 3.75 g/ml in addition to the standard 2.5 g/ml did not show improved agreement by the AP method. While these results do not indicate that the AP method is more accurate than the BACTEC 460TB method, laboratories should not report EMB monoresistance based on BACTEC 460TB results alone. Monoresistance to EMB should only be reported following confirmation by the AP method. Microcolonies could not be confirmed as resistant by the BACTEC 460TB method or by repeat testing with the AP method and do not appear to be indicative of resistance.Radiometric detection of bacterial growth (BACTEC 460TB system; Becton Dickinson and Company, Sparks, Md.) is the most commonly used method in the United States for determining resistance to the primary drugs used to treat Mycobacterium tuberculosis disease (15). This technique was designed to provide rapid susceptibility test results for streptomycin (SM), isoniazid (INH), rifampin (RIF), and ethambutol (EMB) that are equivalent to those obtained by the reference agar proportion (AP) method using Middlebrook agar (7,9,10,12,13).Testing of M. tuberculosis for susceptibility to EMB can be problematic by both the radiometric and AP methods. This may be due to the bacteriostatic nature of EMB, the reduced activity of the drug in a culture medium, or the narrow range between the MICs of susceptible and resistant isolates of M. tuberculosis (4, 6). While the radiometric method has been modified over the years, whether it accurately determines susceptibility to EMB remains in question (5,9,14,16,17). Decisions are unclear on the interpretation and reporting of small colonies of mycobacteria (microcolonies) as resistant mutants on the EMB drug quadrant by AP testing (11). To characterize the extent of these problems with EMB susceptibility testing and provide further information for assistance with test interpretation, we collected and analyzed data from four public health laboratories in a multicenter study. MATERIALS AND METHODSStudy design. Mycobacteriol...

Second-line anti-tuberculosis drug concentrations for susceptibility testing in the MODS assay

“…Studies of the MGIT960 platform have demonstrated sensitivities for detecting resistance as defined by the proportion method for EMB of 63-100%, with sensitivities for detecting STM resistance of 77-100% [5,24,31]. Similar results have been observed for BACTEC 460 (Becton Dickinson) with sensitivities for resistance as defined by the proportion method of 25-91% for EMB and 51-95% for STM [5,12,24,31,32]. Comparison of MODS results for EMB and STM with a more reliable automated DST system may be necessary to validate the minimum inhibitory concentrations found in this study.…”

“…Developed in Peru, MODS has been shown to be a rapid, reliable, low-cost method for DST of isoniazid and rifampicin, but studies have not yet assessed performance in direct DST for second-line drugs [10]. Additionally, data on ethambutol (EMB), streptomycin (STM) and pyrazinamide (PZA) DST in MODS have failed to clearly demonstrate high performance at the drug concentrations employed [10][11][12].…”

Ethambutol Resistance as Determined by Broth Dilution Method Correlates Better than Sequencing Results with embB Mutations in Multidrug-Resistant Mycobacterium tuberculosis Isolates

“…At the genetic level, embB mutations, especially embB306 mutations, are believed to be a major cause of EMB resistance in M. tuberculosis, while several studies revealed that embB mutations had been observed in both EMB-resistant and EMB-susceptible M. tuberculosis strains (5, 9). Because conventional culturebased phenotypic methods of EMB susceptibility testing are notoriously problematic, the presumably inaccurate conventional drug susceptibility results may be the major cause of the "EMBsusceptible" isolates that harbored embB mutations (13,14). Similarly, the problems of conventional proportion susceptibility were most likely responsible for the discrepancies between molecular and phenotypic EMB resistance test results (15).…”

Evaluation of antitubercular drug insertion into preformed dipalmitoylphosphatidylcholine monolayers

“…Ethambutol molecules are acidic in nature and less water soluble than INH molecules [10]. Ethambutol has a bacteriostatic action [21]. It changes the structure of the mycobacterial cell wall, making it more permeable to the other antitubercular drugs [22].…”