Bactec mgit 960

Prior to the DST in BACTEC MGIT 960 system, for purity check, 0.1 μl of each organism suspension were streaked on sterile sheep blood agar plates and incubated at 37 ^OC. Susceptibility testing was then done on pure isolates following the standard operating procedures. Briefly, each PZA drug vial was reconstituted with 2.5 ml of sterile distilled water to make a stock solution of 8000 μg/ml. Two 7 ml MGIT tubes labeled GC (Growth control) and PZA for each isolate were placed in their correct sequence for the 2-tube DST carrier as recommended in the BACTEC MGIT 960 user’s manual. The PZA tubes were inoculated by adding 0.5 μl of the organisms’ suspension and the contents mixed by gently inverting the tubes 4 times. The positive and negative control organisms were prepared as described for the isolates and separately added to the GC and PZA. The final PZA drug concentration was 100 μg/ml. All tubes were then entered into the BACTEC MGIT 960 machine for growth monitoring and interpretation.

Sputum samples submitted for the mycobacterial culture test were inoculated into MGIT broth and cultured using the Bactec MGIT 960 instrument (Becton, Dickinson Cockeysville, MD, USA) (20 ). Positive MGIT broths (flagged positive by the instrument and acid-fast bacillus [AFB] smear was confirmed positive) using the MGIT 960 instrument were subjected to a transcription-reverse transcription concerted reaction (TRC) for the rapid detection of the M. tuberculosis complex by TRCReady-80 (Tosoh Bioscience, Tokyo, Japan) (27 (link)). The detailed methods for TRC are described in the supplemental methods.

Clinical specimens, including sputum, bronchoalveolar lavage fluid, fine-needle aspiration (FNA) tissues, pleural fluid and other body fluids were collected from patients with suspected TB and subjected to cultivation with Bactec MGIT 960 instrument (Becton Dickinson, Cockeysville, MD, USA) in accordance with relevant guidelines, then positive cultures were switched to Lowenstein-Jensen medium (Baso, Zhuhai, China). All the MTB isolates were validated by both the growth test on p-nitrobenzoic acid containing medium (Baso, Zhuhai, China) and MBP 64 antigen detection kit (Genesis, Hangzhou, China). Nontuberculosis mycobacteria (NTM) were excluded.

All material samples suspected of mycobacterial contamination in the Juntendo university hospital were cultured in mycobacteria growth indicator tube (MGIT; Becton Dickinson, USA) broth and incubated at 37 °C in the BACTEC MGIT 960 (Becton Dickinson, USA) instrument with ambient air. MGIT positive tubes were classified as M. abscessus based on the results of DNA–DNA hybridization (DDH) analysis (DDH Mycobacterium “Kyokuto” kit; Kyokuto Pharmaceutical Industrial, Japan) or matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Detected species were reconfirmed as three subspecies of M. abscessus complex by sequencing the 16S rRNA, rpoB, hsp65, and erm genes [20 , 21 (link)]. All strains of M. abscessus were cultured on BD trypticase soy agar II with 5% sheep blood (Blood agar; Nippon Becton-Dickinson and Company, Japan) at 35 °C for approximately 4 to 6 days in an aerobic atmosphere. The study protocol was approved by the Ethics Committee of Juntendo University School of Medicine (no. 18–010 and 19–038).

BACTEC MGIT 960 liquid culture was used. Two milli liter samples were mixed with 4% NaOH solution at 1: 1 ~ 1: 2 and added into a 50 mL centrifuge tube. After shaking and fully liquifying, the samples were left standing for 15 min, and 0.1 mol/L phosphate buffer solution (PBS) was added to 45 mL. After centrifugation at 3,000 × g for 20 min, the supernatant was discarded, 1 mL 0.1 mol/L PBS was added and mixed, 0.5 mL was inoculated into the MGIT 960 culture tube (contains BBL MGIT nutritional supplement OADC and BBL MGIT miscellaneous bacteria inhibitor PANTA) (Becton, Dickinson and Company, United States) and placed in the BACTEC MGIT 960 incubator for 42 days culture. The instrument automatically reports the results.

The quality of sputum was checked upon reception, saliva and soil contaminated specimens were rejected, and participants were asked to bring another specimen. Ziehl-Neelsen (ZN) staining microscopy was done on one early morning and spot specimen for immediate delivery of result. Fine needle aspirate (FNA) samples were also collected from a swollen superficial lymph node of EPTB suspected patients by pathologists. The first few drops of aspirate were placed on a clean slide for FNA cytology and the remaining were sent for culture.
Morning sputum and all other body fluids were processed by NaOH-NALC method for culture. Five hundred microliter processed specimens were inoculated on Mycobacterium Growth Indicator Tube (MGIT) and incubated in the BACTEC MGIT⁹⁶⁰ (Becton Dickinson, USA) system as per manufacturer recommendation [16 ]. By the time the machine flags positive, the tubes were withdrawn from the BACTEC MGIT⁹⁶⁰ system and then were inoculated on blood agar and examined by ZN Microscopy to differentiate contamination from real growth. SD BIOLINE TB Ag MPT64 Rapid test was done for tubes which were positive for acid fast bacilli under microscopy and no growth on blood agar after 48 hrs incubation to verify MTBC. Contaminated samples were reprocessed again and inoculated on liquid media and placed in the MGIT⁹⁶⁰ system.

Following WHO recommendations, the following culture methods were used to determine drug resistance: a modified proportion method on a liquid nutrient medium in a system with automatic growth detection (Bactec MGIT 960 (Becton Dickinson, Sparks, NV, USA)) for first-line drugs (streptomycin, isoniazid, rifampicin, ethambutol, pyrazinamide) and second-line drugs (levofloxacin, moxifloxacin, amikacin, linezolid) and by the method of absolute concentrations on a solid LJ medium for first-line drugs (streptomycin, isoniazid, rifampicin, ethambutol). For the second-line drugs, the absolute concentration method has not been validated but was approved for use [32 ].
Critical concentrations for the proportion method in the Bactec MGIT 960 system were as follows: isoniazid—0.1 mg/L, rifampicin—0.5 mg/L, ethambutol—5.0 mg/L, pyrazinamide—100 mg/L, streptomycin—1.0 mg/L, levofloxacin—1.0 mg/L, moxifloxacin—0.25 mg/L, linezolid—1.0 mg/L, ethambutol—5.0 mg/L, and amikacin—1.0 mg/L. Critical concentrations for the method of absolute concentrations were as follows: isoniazid—1 mg/L, rifampicin—40 mg/L, ethambutol—2 mg/L, streptomycin—10 mg/L, ofloxacin—2 mg/L, capreomycin—30 mg/L, and kanamycin—30 mg/L [29 ].