Laboratory testing

Before processing, direct smears were prepared from each sample for staining with auramine O and observed using a fluorescence microscope (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany).³ These samples were also tested by MPSM (http://www.corebiotech.co.kr). In brief, 2 mL of the remaining sputum samples were treated with Mono-Prep Solution (Core Biotech Inc., Ltd. Gwangju, South Korea) at a ratio of 1:2 for 30 minutes. Subsequently, a microscope slide and a filter tank were placed in the corresponding position of the matching instrument. A volume of 1.5 mL of the sample mixture was transferred into the filter tank. The Mono-Prep system collected bacilli by the dual membrane filtration method (Figure 1), where the upper membrane intercepted the impurities and the lower membrane intercepted Mycobacterium tuberculosis (MTB). The smears were then automatically prepared by the negative pressure adsorption method for the following staining step with auramine O and observed under a fluorescence microscope.

Dual membrane filtration method and auto-smear method (obtained from the instructions of the Mono-Prep system)

For the Xpert MTB/RIF method, the sample reagent was added to the sputum sample at a ratio of 2:1, oscillated for 15 to 30 seconds, and incubated for 15 minutes at room temperature. A volume of 2 mL of the treated sample was transferred into a cartridge. The cartridge was loaded into the Xpert MTB/RIF instrument (GeneXpert; Cepheid Inc., Sunnyvale, CA, USA) and an automatic process completed the remaining assay steps. After the reaction ended, the results could be directly observed under the detection system window.⁴

The remaining sputum sample was then decontaminated and thinned by treating it with 2% sodium hydroxide and 0.5% N-acetyl-L-cysteine for 15 minutes. The sputum was then processed for culture by centrifugation following a standard laboratory protocol.