Mgit 960

The Bactec MGIT 960 assay is an automated assay that is used to evaluate M. tuberculosis growth and that also provides information on PZA susceptibility. The assay was performed according to standard guidelines. Briefly, a loopful of M. tuberculosis culture grown on solid 7H10 medium was suspended in a tube containing beads of 1-mm diameter. This was vortexed and incubated for 20 min, and the bacterial suspension was transferred to a separate tube, where its turbidity was adjusted to a 0.5 McFarland standard equivalent. Dilutions (1:5 and 1:50) were then prepared using saline solution, and 500 μl was added to 7H9 medium supplemented with oleic acid-albumin-dextrose-catalase (OADC), with and without 100 μg/ml PZA. The tubes were incubated in the Bactec MGIT 960 instrument. A final readout representing the ratio between the fluorescences of the strain in the presence and absence of PZA was taken to indicate whether the strain was sensitive or resistant to PZA.

Testing for isoniazid and rifampicin in surveys and continuous surveillance uses either molecular or phenotypic methods. For the 7 countries mentioned, culture isolates from enrolled patients were tested using phenotypic methods according to the most recent WHO recommendations at the time [19 ], by either the Löwenstein-Jensen proportion method (Azerbaijan, Bangladesh, Pakistan, the Philippines, and Ukraine) or MGIT 960 (Becton Dickinson, Sparks, MD, US; Belarus and South Africa) at the following concentrations: 0.2 mg/mL isoniazid and 40.0 mg/L rifampicin on Löwenstein-Jensen medium and 0.1 mg/mL isoniazid and 1.0 mg/L rifampicin on MGIT 960.

All specimens were stained for acid-fast microscopic examination using Ziehl-Neelsen stain, before sample concentration. MTB isolation was performed as described in detail previously [9 (link)]. Briefly, samples were decontaminated with N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH) method and resuspended in 2 ml Phosphate Buffer Saline (PBS): 500 μl were inoculated onto 2 solid slant media (Lowenstein-Jensen; Heipha Diagnostika Biotest, Germany) and 500 μl into liquid culture (MGIT 960; Becton Dickinson, USA). Solid and liquid cultures were considered negative after 42 days of incubation without isolation of any Mycobacteria. Positive cultures were identified as MTB by MGIT TBc Identification Test (Becton Dickinson). Susceptibility of MTB isolates to first-line drugs (Isoniazid, Rifampicin, Ethambutol, Pyrazinamide) was tested by the “gold standard” automatic MGIT 960 system.
“Time to positivity” (TTP) was defined as the number of days from MGIT inoculation to the positive culture result using Epicenter software (Becton Dickinson). An aliquot of 500 μl was tested with Xpert MTB/RIF assay (Xpert, Cepheid, USA) according to manufacturer’s instruction. An aliquot of 500 μl was stored at -20 °C for further use; when culture results were available, only MTB-positive aliquots were archived at -20 °C while the MTB-negative aliquots were discarded.