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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-07-01
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| Series: | Diagnostics |
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| Online Access: | https://www.mdpi.com/2075-4418/12/7/1745 |
| _version_ | 1797406808677023744 |
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| author | Maxim Lebedev Aaron B. Benjamin Kent J. Koster Kathryn E. Broyles Sathish Kumar Joseph M. Jilka Jeffrey D. Cirillo |
| author_facet | Maxim Lebedev Aaron B. Benjamin Kent J. Koster Kathryn E. Broyles Sathish Kumar Joseph M. Jilka Jeffrey D. Cirillo |
| author_sort | Maxim Lebedev |
| collection | DOAJ |
| description | 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 <i>Mycobacterium tuberculosis</i> 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. |
| first_indexed | 2024-03-09T03:31:57Z |
| format | Article |
| id | doaj.art-eb98b19203bf4f4faa318b2042817fcf |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-03-09T03:31:57Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Diagnostics |
| spelling | doaj.art-eb98b19203bf4f4faa318b2042817fcf2023-12-03T14:55:26ZengMDPI AGDiagnostics2075-44182022-07-01127174510.3390/diagnostics12071745Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of CareMaxim Lebedev0Aaron B. Benjamin1Kent J. Koster2Kathryn E. Broyles3Sathish Kumar4Joseph M. Jilka5Jeffrey D. Cirillo6Department of Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, TX 77807, USADepartment of Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, TX 77807, USADepartment of Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, TX 77807, USADepartment of Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, TX 77807, USADepartment of Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, TX 77807, USAPulmonescence Diagnostics, Inc., Temple, TX 76504, USADepartment of Microbial Pathogenesis and Immunology, Texas A&M School of Medicine, Bryan, TX 77807, USATuberculosis 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 <i>Mycobacterium tuberculosis</i> 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.https://www.mdpi.com/2075-4418/12/7/1745tuberculosisdiagnosticsbeta lactamaseBlaClactosemannitol |
| spellingShingle | Maxim Lebedev Aaron B. Benjamin Kent J. Koster Kathryn E. Broyles Sathish Kumar Joseph M. Jilka Jeffrey D. Cirillo Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of Care Diagnostics tuberculosis diagnostics beta lactamase BlaC lactose mannitol |
| title | Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of Care |
| title_full | Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of Care |
| title_fullStr | Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of Care |
| title_full_unstemmed | Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of Care |
| title_short | Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb) Diagnostic Reagents for Use at the Point of Care |
| title_sort | stabilization of tuberculosis reporter enzyme fluorescence reftb diagnostic reagents for use at the point of care |
| topic | tuberculosis diagnostics beta lactamase BlaC lactose mannitol |
| url | https://www.mdpi.com/2075-4418/12/7/1745 |
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