No abstract

The BACTEC MGIT 960 system, a fully automated, nonradiometric, noninvasive system for detection and drug susceptibility testing of mycobacteria, was evaluated for the ability to test susceptibilities to second-line drugs. In this study, which was carried out in three phases (phase I, mostly susceptible strains; phase II, mostly resistant strains; phase III, final testing of the optimal drug concentrations found in phases I and II), we established the critical concentrations for seven drugs to be tested in the BACTEC MGIT 960 system compared to the BACTEC 460TB system. The critical concentrations for the seven drugs used in the MGIT 960 system are as follows: amikacin, 1.0 g/ml; capreomycin, 2.5 g/ml; ethionamide, 5.0 g/ml; protionamide, 2.5 g/ml; ofloxacin, 2.0 g/ml; rifabutin, 0.5 g/ml; linezolid, 1.0 g/ml. Our results demonstrate that the BACTEC MGIT 960 system is an accurate method for rapid testing of the susceptibilities of Mycobacterium tuberculosis to second-line drugs.Drug susceptibility testing (DST) for both primary and secondary antituberculosis drugs with the broth-based radiometric BACTEC 460 TB system (Becton Dickinson Diagnostic Systems, Sparks, MD) is well established and is considered the "gold standard" (15). However, due to increasing concern about the use and disposal of radioactive material, there is a rapid trend toward using commercially available nonradiometric broth-based culture and susceptibility testing methods. BACTEC MGIT 960 (Becton Dickinson Diagnostic Systems) is a new nonradiometric system which is considered equivalent to the BACTEC 460 in performance. Recovery of mycobacteria from clinical specimens as well as DST for first-line drugs has been thoroughly studied for the MGIT 960 system (3,4,5,7,8,10,11,12). However, no thorough multicenter study has been carried out establishing DST for second-line and newer drugs currently being used in the treatment of tuberculosis. According to the WHO reports, global drug resistance is an increasing concern (18). Some countries are reporting high resistance even against second-line drugs (1, 17). Therefore, it is important that nonradiometric broth-based systems should also offer DST procedures for drugs other than those considered first-line.The primary aim of this multicenter study was to develop a basic protocol, establish critical test concentrations for seven second-line and newer drugs, including a few that have been introduced recently, and then test a large number of clinical isolates. For comparison, BACTEC 460 was used as the gold standard, since critical test concentrations of most of the drugs have already been established for this system (9). It is anticipated that this study will provide a guideline for rapid brothbased susceptibility testing not only of the drugs that have been included here but also of other drugs that are used in the treatment of tuberculosis or will be introduced in the near future. MATERIALS AND METHODSStudy sites. This study was carried out at three sites: (i) the National Reference Center for Mycobacte...

Several subtypes of Mycobacterium kansasii have been described, but their respective pathogenic roles are not clear. This study investigated the distribution of subtypes and the pathogenicity of M. kansasii strains (n ‫؍‬ 191) isolated in Switzerland between 1991 and 1997. Demographic, clinical, and microbiological information was recorded from clinical files. Patients were classified as having an infection according to the criteria of the American Thoracic Society. Subtypes were defined by PCR-restriction enzyme analysis of the hsp65 gene. Subtype 1 comprised 67% of the isolates (n ‫؍‬ 128), while subtypes 2 and 3 comprised 21% (n ‫؍‬ 40) and 8% (n ‫؍‬ 15), respectively. Other subtypes (subtypes 4 and 6 and a new subtype, 7) were recovered from only 4% of patients (n ‫؍‬ 8). M. kansasii subtype 1 was considered pathogenic in 81% of patients, while M. kansasii subtype 2 was considered pathogenic in 67% of patients and other subtypes were considered pathogenic in 6% of patients. The majority of patients with M. kansasii subtype 2 were immunocompromised due to the use of corticosteroids (21% of patients) or coinfection with HIV (62.5% of patients). Subtyping M. kansasii may improve clinical management by distinguishing pathogenic from nonpathogenic subtypes.

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536 T , M. massiliense CIP 108297 T , and M. bolletii CIP 108541 T ) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Drug-resistant strains of Mycobacterium tuberculosis, though not a novel phenomenon, are emerging worldwide. According to the latest figures of the World Health Organization and the International Union against Tuberculosis and Lung Diseases, drug resistance, in particular acquired resistance, has poured additional fuel on the fire of global tuberculosis (TB) (18). Several outbreaks of multidrug-resistant TB (7) were characterized by delayed diagnoses, inadequate treatment regimens, high rates of mortality, and significant rates of transmission and have taught us two lessons: first, the days are definitely gone where full susceptibility of TB bacilli to front-line drugs can be taken for granted. Second, rapid detection of drug resistance is paramount, not only for effective treatment of TB patients but also for initiating adequate public health measures.In the quest for new nonradiometric, culture-based strategies which allow both rapid detection of acid-fast bacilli and testing of susceptibility to antimicrobial agents, new liquid medium-based systems, such as the MB/BacT (Organon-Teknika, Durham, N.C.), ESP Culture System II (AccuMed International, Westlake, Ohio), MB Redox (Biotest, Dreieich, Germany), and the Mycobacteria Growth Indicator Tube 960 (MGIT 960, Becton Dickinson Microbiology Systems, Sparks, Md.), have become available. They all aim not only at recovering mycobacteria from clinical specimens but also at generating antimicrobial susceptibility testing (AST) data with a shorter turnaround time than that observed with the current "gold standard," the agar proportion method (11). The performance of a new system should be comparable with that of the BACTEC 460 TB system, with elimination of the two core problems associated with the old BACTEC 460 TB technology, i.e., the risk of needle punctures and disposal of radioactive waste. Preliminary studies utilizing those new systems report good overall agreement of AST results with those generated with established methods (1, 3-5, 8, 10, 12-13, 15).Recent automation of the MGIT 960 technology was another step forward, as it allows continuous monitoring of positive fluorescence, which is based on bacterial growth. It is noninvasive and eliminates potential reading difficulties during visual judging of the tubes, apart from saving labor. The threshold algorithms help in determining the susceptibility automatically.In this multicenter study we have evaluated the reproducibility and reliability of the BACTEC MGIT 960 instrument for testing of M. tuberculosis susceptibility to isoniazid (INH), rifampin (RIF) ethambutol (EMB), and streptomycin (STR) and have compared the results to those obtained by the radiometric procedure. Discordant results were resolved by testing the strains with the agar proportion method using Löwenstein-Jensen (LJ) medium (6). This was done by an additional site which thus acted as an independent arbiter. Last, in order to address safety, we performed drug susceptibility testing in plastic MGITs, in addition to the glass tubes.

No abstract

In a multicenter study involving three reference centers for mycobacteria, the rate of recovery of acid-fast bacilli (AFB) and the mean time to their detection from clinical specimens was determined by using the Mycobacteria Growth Indicator Tube (MGIT). These parameters were compared to those assessed by the radiometric BACTEC 460 TB system and by cultivation on solid media. Clinical specimens (n ‫؍‬ 1,500) were pretreated with N-acetyl-L-cysteine (NALC)-NaOH. The contamination rates for MGITs were 2.0% (center 1), 13.8% (center 2), and 6.1% (center 3). A total of 180 mycobacterial isolates were detected (M. tuberculosis complex, n ‫؍‬ 113; nontuberculous mycobacteria [NTM], n ‫؍‬ 67). When using a combination of liquid and solid media (the current "gold standard" for culture), MGIT plus solid media detected 156 (86.7%) of the isolates, whereas BACTEC plus solid media recovered 168 (93.3%) of all AFB. Between these two gold standards there was no statistically significant difference (P > 0.05). The combination of MGIT plus BACTEC detected 171 (95.0%) of all isolates (compared with MGIT plus solid media, P < 0.01; compared with BACTEC plus solid media, P > 0.05). Considering the efficacies of the different media separately, MGIT was superior to solid media (although not significantly; P > 0.05) in detecting AFB but was inferior to the BACTEC system (P < 0.01). The mean time to the detection of M. tuberculosis complex was 9.9 days with MGIT, 9.7 days with BACTEC, and 20.2 days with solid media. NTM needed, on average, 11.9, 13.0, and 22.2 days to appear by the three methods, respectively. In conclusion, MGIT proved to be a valuable alternative to the radiometric cultivation system.

Background-Electrophysiological cardiac devices are increasingly used. The frequency of subclinical infection is unknown. We investigated all explanted devices using sonication, a method for detection of microbial biofilms on foreign bodies. Methods and Results-Consecutive patients in whom cardiac pacemakers and implantable cardioverter/defibrillators were removed at our institution between October 2007 and December 2008 were prospectively included. Devices (generator and/or leads) were aseptically removed and sonicated, and the resulting sonication fluid was cultured. In parallel, conventional swabs of the generator pouch were performed. A total of 121 removed devices (68 pacemakers, 53 implantable cardioverter/defibrillators) were included. The reasons for removal were insufficient battery charge (nϭ102), device upgrading (nϭ9), device dysfunction (nϭ4), or infection (nϭ6). In 115 episodes (95%) without clinical evidence of infection, 44 (38%) grew bacteria in sonication fluid, including Propionibacterium acnes (nϭ27), coagulase-negative staphylococci (nϭ11), Gram-positive anaerobe cocci (nϭ3), Gram-positive anaerobe rods (nϭ1), Gram-negative rods (nϭ1), and mixed bacteria (nϭ1). In 21 of 44 sonication-positive episodes, bacterial counts were significant (Ն10 colony-forming units/mL of sonication fluid). In 26 sterilized controls, sonication cultures remained negative in 25 cases (96%). In 112 cases without clinical infection, conventional swab cultures were performed: 30 cultures (27%) were positive, and 18 (60%) were concordant with sonication fluid cultures. Six devices and leads were removed because of infection, growing Staphylococcus aureus, Streptococcus mitis, and coagulase-negative staphylococci in 6 sonication fluid cultures and 4 conventional swab cultures. Conclusions-Bacteria can colonize cardiac electrophysiological devices without clinical signs of infection. (Circulation.