Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of Care.

Tuberculosis is one of the most frequent causes of death in humans worldwide. One of the primary reasons tuberculosis remains a public health threat is that diagnosis can take weeks to months, is often not very sensitive and cannot be accomplished in many remote environments. A rapid, sensitive and inexpensive point-of-care (POC) diagnostic would have a major impact on tuberculosis eradication efforts. The tuberculosis diagnostic system REFtb is based on specific detection of the constitutively expressed β-lactamase (BlaC) in Mycobacterium tuberculosis using a custom fluorogenic substrate designated as CDG-3. REFtb has potential as a diagnostic for tuberculosis that could be very inexpensive (<USD 2.00/test), used at the POC and could provide definitive diagnosis within 10 min. However, the reagents for REFtb are currently in liquid form, making them more susceptible to degradation and difficult to transport. We evaluated the improvement in the stability of REFtb reagents by lyophilization under a variety of conditions through their effects on the performance of REFtb. We found that lyophilization of REFtb components produces an easily reconstituted powder that displays similar performance to the liquid system and that lactose represents one of the most promising excipients for use in a final POC REFtb diagnostic system. These studies provide the foundation for the production of a stable POC REFtb system that could be easily distributed worldwide with minimal or no requirement for refrigeration.