C-2017: Cryopreservation of Mycobacterium tuberculosis (MTB) complex cells and sputum specimens for MTB diagnosis

Abstract

Successful long-term preservation of Mycobacterium tuberculosis (MTB) cells or sputum specimens is very important for sample transport, biobanking, research and the development of new drugs, vaccines, biomarkers, and diagnostics. In this work, M . bovis Bacillus Calmette–Guerin (BCG) and M. tuberculosis H37Ra were used as models of MTB complex strains to study cryopreservation of MTB complex cells in diverse sample matrices (phosphate buffer saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum). Efficacy of cryopreservation was quantified by microbiological culture. Results showed that among the variables tested, slow cooling rate was the most critical factor for the successful cryopreservation of Mycobacterium tuberculosis complex cells. Preincubation of frozen/thawed MTB cells in 7H9 broth before culturing did not help the cells repair cryoinjury. Cell inactivation by fast cooling was not associated with a compromised cell envelope, as indicated by the comparison results of microbiological culture and the BacLight live/dead staining. After cryopreservation, the MTB cell surface antigen can still function well for the MTB diagnosis as biomarker. For the Point-of-Care (POC) diagnosis of MTB, an occupationally safe (biosafe) sputum liquefaction protocol and a semi-automated antibody-based microtip immunofluorescence sensor were developed. Sputum was treated with a synergistic chemical–thermal protocol that included moderate concentrations of NaOH and detergent at 60 °C for 5–10 min. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. The biosafe sputum liquefaction method, cryopreservation protocol, and the microtip device make it possible to diagnose the tuberculosis with high feasibility, sensitivity and accuracy.