Genotype mycobacterium cm kit

The processing of PW samples was carried out for semiquantitative assessment (acid-fast bacilli (AFB) smear) and isolation purposes for all cases using the NaOH-NALC method¹³ . Briefly, 1 ml of PW was mixed with an equal volume of 2% NaOH-NALC solution (consisting of 4% sodium hydroxide, 2.9% sodium citrate and 0.5% N-acetyl L cysteine) using a vortex mixer and incubated at room temperature for 15 min. Subsequently, 40 ml of sterile 0.067 M phosphate buffer (pH 7.0) were added, mixed by inverting 3–4 times, and centrifuged at 3000 × g for 20 min at 4 °C. The supernatant was then discarded and the pellets were resuspended with 1 ml of sterile 0.067 M phosphate buffer. Samples of 0.1 ml, 0.5 ml and 1 drop of the resuspended samples were added to Löwenstein Jensen Medium (LJ medium), MGIT media, and slides for AFB staining, respectively. Culture medium was incubated at 37 °C for four to eight weeks. AFB staining was performed using the Kinyoun technique in both sediment-positive and culture-positive samples. In-house multiplex PCR method^{14 (link)} and/or the GenoType Mycobacterium CM kit (Hain Life Science, Germany) were used to test AFB-positive colonies for identification of the presence of Mtb.