Mgit 960 system

Clinical, microbiological, and radiographic information were collected retrospectively. Cultures were grown in both solid Ogawa media and the BACTEC MGIT 960 system, and isolated NTM were identified into species [4 (link)]. Identification was performed based on sequence analysis of the 16S rRNA and rpoB gene using the algorithm described in the Clinical and Laboratory Standards Institute guidelines MM18-A [12 ]. Once the NTM was identified as MAC, a drug susceptibility test for clarithromycin was performed. The minimal inhibitory concentration (MIC) test was performed at the Korean Institute of Tuberculosis, and was determined using the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute guidelines [12 ]. Isolates were considered susceptible if the MIC of clarithromycin was ≤8 μg/mL, resistant if ≥32 μg/mL, and intermediate if 16 μg/mL on Mueller–Hinton agar. Chest computed tomography findings were categorized as the nodular bronchiectatic form when bilateral bronchiectasis and cellular bronchiolitis were mainly present, and the upper lobe cavitary form when cavities in the upper lobes were observed [13 (link)].

All MTBC isolates underwent indirect phenotypic first-line DST using the liquid BACTEC MGIT 960 system, according to the manufacturer’s recommendations [45 ,46 ]. First-line anti-TB agents tested for resistance included RIF, INH, ethambutol (EMB) and pyrazinamide (PZA). Susceptibilities were tested at the following standard critical concentrations: RIF, 1.0 μg/ml; INH, 0.1 μg/ml and 0.4 μg/ml; EMB, 5.0 μg/ml; PZA, 100 μg/ml.[6 (link)] PSQ was performed by the CDPH MDL using the PyroMark Q96ID system (Qiagen, Valencia, CA) to sequence molecular targets associated with resistance to RIF, INH, fluoroquinolones and anti-TB injectable agents, as has been previously described [14 (link),47 (link),48 (link)].

In the present study, adult DR-TB patients in the Netherlands were our population of interest. We included adult patients 18 years and older who were diagnosed with tuberculosis disease during the period 2005–2015, caused by M. tb pathogen proven to be resistant to at least one of the first-line antituberculosis drugs. A phenotypical confirmation test has been used as a standard test in the Netherlands between 2005 and 2007. However, a combination test, i.e., phenotypic test (Bactec MGIT 960 system) and genotypic test (Genotype MTBDR plus assay or Line Probe Assay (LPA)), have been applied since 2007. Drug susceptibility testing (DST) was conducted to determine resistance to first-line anti-tuberculosis drugs. If the resistance had been confirmed, the DST was extended to the second-line drugs, except for isoniazid, pyrazinamide and ethambutol mono-resistance. We retrospectively followed-up up to 24 months for patients identified as DR-TB. The observation was started from the time the diagnosis of DR-TB was made to the outcome of TB treatment was reported. Patients who had not started treatment and had an unknown treatment outcome were excluded from the analysis for the incidence and patient risk factors for poor outcome of TB treatment. Moreover, patients who had an unknown treatment outcome were included for further analysis of a not-evaluated patient outcome.

Five ml of sputum was collected to a 50 ml centrifuge tube with the addition of an equal amount of 2% NALC-NaOH pretreatment liquid. The tube was vortexed for 20 s and left to react for 15 min. With an addition of 50 ml of sterile PBS, the pretreated solution was centrifuged at 3000 g for 15 min, and then, the supernatant was discarded. Thereafter, the solution was added with 2 ml of PBS and mixed. Next, 0.5 ml of the mixture was inoculated in a MGIT culture tube and incubated using the BACTEC MGIT 960 system. If the sample is positive, the red indicating light of the instrument will be on immediately along with an alarm sound.