21st European Congress of Clinical Microbiology and Infectious Diseases

Bead-based specimen concentration for mycobacterial culture

Abstract number: P2023

Mitarai S., Yamada H., Mizuno K., Chikamatsu K., Aono A., Sugamoto T., Karinaga R., Hatano T.

Objectives: The centrifugation is a hazardous laboratory process. Genetein Co. Ltd. has recently developed a beads-based M. tuberculosis collection and concentration method, named TRICORE, to avoid centrifugation process. The efficiency of TRICORE for the concentration of M. tuberculosis from the sputum was evaluated.

Methods: A total of 30 sputum specimens were collected from the patients with active tuberculosis. The specimens were mixed well with same volume of NALC-NaOH for the digestion and decontamination, and incubated at room temperature for 15 min followed by neutralisation with phosphate buffer (PB: pH 6.8). To obtain various degrees of smear positivity, 5ml of neutralised specimen was dispensed into two tubes, and 0.5ml of the original was serially diluted with 4.5ml of PB into by 1/10 and 1/100. A total of 180 pre-treated specimens (30 specimens × 3 dilutions, × 2 series) were prepared for the following experiments. One series of specimens were centrifuged at 3,000g for 20 min at 4°C. The supernatant was discarded and the sediment was re-suspended with 1ml of PB. For the TRICORE method, 200 ml of bacteria capture beads, 100 ml of capture sol, and 100 ml of co-precipitation beads were added to each tube, and mixed by inversion rotation for 3 minutes. After 3 minutes' incubation, the supernatant was removed remaining 1 ml of the specimen attracting the co-precipitated beads by magnet. 100 ml and 500 ml of each residue were inoculated into 2% Ogawa and MGIT (Becton Dickinson) media, respectively.

Results: Among 90 pre-treated sputum specimens, 51 (57.3%) and 55 (61.8%) strains were recovered by MGIT system with the centrifugation and the TRICORE method, respectively (Chi square test: p = 0.5413). The time to detection for centrifugation method was 359.3±117.0 hours, while that of TRICORE method was 377.6±162.3 (p = 0.5637). However, the numbers of colonies recovered on solid media were significantly higher in the TRICORE method (p = 0.003). Especially, among the smear-negative specimens, the culture positivity of TRICORE method was 39.6%, while the centrifugation method was 15.1% (p = 0.005).

Conclusion: The TRICORE is comparable to the conventional centrifugation method, and considered to collect the tubercle bacilli more efficiently than the centrifugation method especially in paucibacillary specimens. The TRICORE, a new concentration method without using centrifugation, will be safe and could yield more positive culture results.