Semi-Automated, Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of Mycobacterium Cells in Sputum: e86018

• Published in: PloS one 2014-01, Vol.9 (1)
• Main Authors: Inoue, Shinnosuke, Becker, Annie L, Kim, Jong-Hoon, Shu, Zhiquan, Soelberg, Scott D, Weigel, Kris M, Hiraiwa, Morgan, Cairns, Andrew, Lee, Hyun-Boo, Furlong, Clement E
• Format: Article
• Language: English
• Subjects: Mycobacterium tuberculosis
• ISSN: 1932-6203
• DOI: 10.1371/journal.pone.0086018

An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60 degree C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 106-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.