e Multidrug therapy is a standard practice when treating infections by nontuberculous mycobacteria (NTM), but few treatment options exist. We conducted this study to define the drug-drug interaction between clofazimine and both amikacin and clarithromycin and its contribution to NTM treatment. Mycobacterium abscessus and Mycobacterium avium type strains were used. Time-kill assays for clofazimine alone and combined with amikacin or clarithromycin were performed at concentrations of 0.25؋ to 2؋ MIC. Phar- Multidrug therapy is a standard practice when treating mycobacterial infections. However, the pharmacodynamic (PD) interactions among the combined drugs are largely unknown. Understanding these interactions will help to identify synergistic combinations with increased antibacterial killing, which ultimately can result in a better treatment outcome.One of the promising combinations is amikacin and clofazimine, given the key role of amikacin in the treatment of NTM infections (2, 3) and the unique characteristics of clofazimine, like its prolonged half-life, its preferential accumulation inside macrophages (4), and the recently found bactericidal activity only after 2 weeks of treatment in the mouse model of tuberculosis (5).Clarithromycin, on the other hand, has substantial in vitro and clinical activity against Mycobacterium avium complex (MAC), and it has been long considered the cornerstone for Mycobacterium abscessus treatment (3).Hence, the examination of its interaction with clofazimine is interesting.Previous studies showed in vitro synergy between clofazimine and amikacin against both rapidly and slowly growing NTM (1, 6). The combination clarithromycin-clofazimine also showed synergy against MAC strains in checkerboard evaluation (7). These checkerboard titrations offer no information on the mechanism of synergistic activity, the exact killing activity of these combinations, or its concentration dependence. We therefore investigated the pharmacodynamic interactions between clofazimine and amikacin, and clofazimine and clarithromycin, against two key NTM species, using time-kill assays analyzed with two pharmacodynamic drug interaction models: the response surface model of Bliss independence (RSBI) and isobolographic analysis of Loewe additivity (ISLA) (8, 9). MATERIALS AND METHODS Bacterial

In a multiple-dose-ranging trial, we previously evaluated higher doses of rifampin in patients for 2 weeks. The objectives of the current study were to administer higher doses of rifampin for a longer period to compare the pharmacokinetics, safety/tolerability, and bacteriological activity of such regimens. In a double-blind, randomized, placebo-controlled, phase II clinical trial, 150 Tanzanian patients with tuberculosis (TB) were randomized to receive either 600 mg (approximately 10 mg/kg of body weight), 900 mg, or 1,200 mg rifampin combined with standard doses of isoniazid, pyrazinamide, and ethambutol administered daily for 2 months. Intensive pharmacokinetic sampling occurred in 63 patients after 6 weeks of treatment, and safety/tolerability was assessed. The bacteriological response was assessed by culture conversion in liquid and solid media. Geometric mean total exposures (area under the concentration-versus-time curve up to 24 h after the dose) were 24.6, 50.8, and 76.1 mg · h/liter in the 600-mg, 900-mg, and 1,200-mg groups, respectively, reflecting a nonlinear increase in exposure with the dose ( < 0.001). Grade 3 adverse events occurred in only 2 patients in the 600-mg arm, 4 patients in the 900-mg arm, and 5 patients in the 1,200-mg arm. No significant differences in the bacteriological response were observed. Higher daily doses of rifampin (900 and 1,200 mg) resulted in a more than proportional increase in rifampin exposure in plasma and were safe and well tolerated when combined with other first-line anti-TB drugs for 2 months, but they did not result in improved bacteriological responses in patients with pulmonary TB. These findings have warranted evaluation of even higher doses of rifampin in follow-up trials. (This study has been registered at ClinicalTrials.gov under identifier NCT00760149.).

The total effect observed for all antibiotics was low and primarily determined by the Emax and not by the Hill's slope. The limited activity detected fits well with the poor outcome of antibiotic treatment for disease caused by RGM, particularly for M. abscessus. An evaluation of drug combinations will be the next step in understanding and improving current treatment standards.

We have assessed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification (Bruker) of nontuberculous mycobacteria from newly positive liquid cultures of respiratory samples. Twelve (22%) of 54 isolates were identified directly from liquid medium. After subculture and with manual laser operation, this rose to 49/54 isolates (91%). MALDI-TOF MS is less promising than previously suggested. N ontuberculous mycobacteria (NTM) are increasingly recognized as mostly opportunistic pathogens of humans. The most frequent disease manifestation is a chronic pulmonary infection (1). Since NTM are environmental microorganisms, their presence in pulmonary samples need not indicate disease per se. The clinical relevance of NTM isolation differs strongly by species (2). Hence, correct identification is of paramount importance (3).Identification of NTM is done mostly by using molecular tools. While accurate, these require a good laboratory infrastructure and trained personnel and are costly. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized identification in general bacteriology and has also been tried for NTM (3). Most studies of MALDI-TOF MS identification of NTM have applied it to pure cultures from strain collections (4) rather than new positive cultures from clinical samples from nonsterile sites.We assessed MALDI-TOF MS identification of NTM directly from new positive liquid cultures of respiratory samples in a mycobacterial disease reference clinic.Primary cultures of respiratory samples were performed with the Mycobacterium Growth Indicator Tube (MGIT) system (BD Bioscience, Erembodegem, Belgium), as well as on Löwenstein-Jensen slants, both at 37°C, after decontamination by the 1% Nacetyl-L-cysteine-sodium hydroxide method (3).All of the positive cultures that we obtained during the MarchApril 2014 and January-February 2015 periods that tested negative in the TBc-ID immunochromatography assay (BD Bioscience) were studied.We performed MALDI-TOF MS identification with the MALDI Biotyper (Bruker Daltonics, Bremen, Germany) platform. Protein extraction from liquid MGIT medium for MALDI-TOF MS analysis was performed by the manufacturer's MycoEx protocol. In short, we collected biomass by aspirating 1.2 ml of liquid medium from the bottom of the MGIT tube and transferring it to a reaction tube, which was then centrifuged for 2 min at 13,000 rpm (8,124 ϫ g; 43-mm rotor radius); the supernatant was pipetted off, the pellet was resuspended in 300 l of high-performance liquid chromatography grade water, and cells were then inactivated by boiling for 30 min. A 900-l volume of pure cold ethanol was then added, and the tube was vortexed and centrifuged for 2 min at 13,000 rpm. The supernatant was pipetted off. The last two steps were repeated once. The pellet was dried at room temperature before the addition of zirconium beads and 10 to 50 l of pure acetonitrile, depending on the pellet size. After 1 min of vortexing, we adde...

The killing effect of amikacin and clarithromycin on M. avium subspecies hominissuis was low, although amikacin activity was higher than that of clarithromycin, supporting its role in a combined therapy. Clarithromycin and moxifloxacin may have similar activity within treatment regimens for M. xenopi disease. Future studies of in vitro and in vivo pharmacokinetic/pharmacodynamic interactions are needed to improve the current regimens to treat these two important slowly growing mycobacteria in pulmonary disease.