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.
“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.
Free full text: Click here
