No 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...

During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 5 1 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16s rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjecturn, a pathogen that resembles the ilonpathogen Mycobacterium gurdonae phenotypically, We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.Approximately 17 years ago the International Working Group on Mycobacterial Taxonomy (IWGMT) began a cooperative open-ended study in which phenotypically unusual strains of s l o~l y growing mycobacteria were collected on a continuing basis. These strains were distributed to participants in the open-ended study for characterization by a broad range of predominantly phenotypic tests, and at intervals the data obtained were subjected to numerical taxonomic (NT) analyses (29-32). The purpose of studying an expanding set of cultures was to characterize slowly growing mycobacterial strains that either represented uncommonly encountered species that had not been represented in previous cooperative studies (10, 25, 28) or belonged to clusters of previously unrecognized taxa. These analyses yielded expanded phenotypic characterizations of members of some clusters that had not been thoroughly characterized before, such as the clusters that included the type strains of Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium asiaticum, and Mycobacterium malmoense (3 1). However, some individual strains and phenotypic clusters emerged that exhibited no unequivocal aff...