Sensitive Detection of Tuberculosis (TB)
Donald Catanzaro (PI) Research Assistant Professor, Department of Biological Sciences
Yuchun Du (Co-PI) Associate Professor, Department of Biological Sciences
Jingyi Chen (Co-PI) Associate Professor, Department of Chemistry and Biochemistry
Funding: Arkansas Biosciences Institute (ABI)
Background and Specific Aims Tuberculosis (TB) is currently the world’s leading cause of death from infectious disease, causing over 1.7 million deaths every year.1 According to the World Health Organization (WHO), in addition to the over 10 million new cases of TB in 2016, there are nearly 2 billion people globally are infected with Mycobacterium tuberculosis (Mtb), the causative agent of TB. Although these latently infected individuals are not ill and cannot transmit TB disease, they are at a higher risk for progression to active TB. While the TB incidence in the United States is low (~3/100,000), to achieve TB elimination in the United States by 2100, the Centers for Disease Control estimates annual percent decline in TB incidence must double from 1.8% per year to 3.9% per year2 which will require significant investment in new TB diagnostics. Rapid diagnosis of TB is the cornerstone of the public health response to TB and at present sputum-smear microscopy is the world’s most popular TB diagnostic test despite its sensitivity of only 20-80% in high burden settings and a false positive rate of up to 40% in low burden settings.1,3,4 The WHO stated in 2014 that the most urgent diagnostic need for TB is an accurate, rapid, non-sputum screening TB test for use on readily accessible clinical samples, such as saliva, blood or urine.5 The primary obstacle to achieving an accurate biomarker assay for TB has been our inability to reliably detect Mtb-specific molecules, such as protein or DNA, in serum from TB patients. As a result of these complications, numerous efforts have been directed toward measuring related/alternative biomarkers of TB (e.g. serodiagnostic tests,6 Interferon Gamma Release Assays,7 and LAM-based assays8) with varying success.
Instrumentation improvements in mass spectrometry (MS) in the past two decades have resulted in high sensitivity, high specificity, and high throughput analysis of biological materials, including powerful methods for analysis of complex biological samples such as total cell lysate or serum. Modern sensitive normal mass spectrometers exhibit: (1) superior sensitivity/specificity to state-of-the-art immunoassays, (2) can detect protein/peptide at sub-attomole levels, and (3) are currently in clinical use to speciate mycobacterial infections.9,10 Similarly, DNA/RNA sequencing/detection technologies have advanced to a degree that a single cell can be analyzed/detected.11,12 For example, it is now possible to analyze global gene expression in a single cell using as little as 10 picograms of total RNAs.13,14
The peptide 10-kDa culture filtrate protein (CFP-10) and 6-kDa early secretory antigenic target (ESAT-6) are well-known Mtb proteins secreted by actively growing Mtb and are currently measured indirectly in commercially available TB tests.7 Recently, our collaborator on our new NIH grant (R01AI137681), Dr. Ye Hu (Arizona State University), developed a prototype NanoDisk-MS system, which use antibody-conjugated nanodisks to trap low levels of TB proteins from TB sera, and use matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) to directly detect tryptic peptides of Mtb proteins trapped on the NanoDisks.15 There is a need to improve this method (test sensitivity and specificity of ~92% and ~96%16,17) in order to meet the WHO performance criteria for TB screening (sensitivity:95%, specificity:90%) and/or TB diagnosis (sensitivity:98%, specificity:98%).
Our central hypothesis is that the precise levels of Mtb peptides or Mtb DNAs can be quantified in patient sera and used to differentiate patients with active TB from those with latent TB infection (LTBI), NTM, non-TB pulmonary disease, and healthy controls. The objective of this application is to couple nanomaterials to mass spectrometry or novel molecular techniques to reliably detect TB molecular markers from patient’s blood. We propose the following two Specific Aims:
Specific Aim 1 is to detect Mtb CFP-10 and ESAT-6 peptides using ultra-sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) coupled with nanoparticles conjugated with Mtb specific antibody.
Specific Aim 2 is to detect Mtb DNAs from serum using Mtb probe-conjugated nanoparticles and PCR.
References
1. World Health Organization (WHO). Global Tuberculosis Report 2017. Geneva, Switzerland: World Health Organization (WHO); 2017.
Stewart RJ, Tsang CA, Pratt RH, Price SF, Langer AJ. Tuberculosis – United States, 2017. MMWR Morb Mortal Wkly Rep. 2018;67(11):317-23. doi: 10.15585/mmwr.mm6711a2. PubMed PMID: 29565838; PubMed Central PMCID: PMC5868206.
2. World Health Organization (WHO). Same-Day Diagnosis of Tuberculosis by Microscopy: WHO Policy Statement. Geneva. 2011. Available from: http://www.ncbi.nlm.nih.gov/pubmed/23586121.
3. World Health Organization (WHO). Systematic screening for active tuberculosis : principles and recommendations [text]. Geneva, Switzerland. 2013.
4. World Health Organization (WHO). High-priority target product profiles for new tuberculosis diagnostics: report of a consensus meeting. Geneva, Switzerland: World Health Organization (WHO); 2014. Available from: http://apps.who.int/iris/bitstream/10665/135617/1/WHO_HTM_TB_2014.18_eng.pdf?ua=1&ua=1.
5. World Health Organization (WHO). Commercial Serodiagnostic Tests for Diagnosis of Tuberculosis: Policy Statement. Geneva, Switzerland: World Health Organization (WHO); 2011.
6. Diel R, Goletti D, Ferrara G, Bothamley G, Cirillo D, Kampmann B, Lange C, Losi M, Markova R, Migliori GB, Nienhaus A, Ruhwald M, Wagner D, Zellweger JP, Huitric E, Sandgren A, Manissero D. Interferon-gamma release assays for the diagnosis of latent Mycobacterium tuberculosis infection: a systematic review and meta-analysis. Eur Respir J. 2011;37(1):88-99. doi: 10.1183/09031936.00115110. PubMed PMID: 21030451.
7. World Health Organization (WHO). The use of lateral flow urine lipoarabinomannan assay (LF-LAM) for the diagnosis and screening of active tuberculosis in people living with HIV: Policy Guidance. Geneva, Switzerland: World Health Organization; 2015.
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15. Liu C, Lyon CJ, Bu Y, Deng Z, Walters E, Li Y, Zhang L, Hesseling AC, Graviss EA, Hu Y. Clinical Evaluation of a Blood Assay to Diagnose Paucibacillary Tuberculosis Via Bacterial Antigens. Clinical chemistry. 2018. doi: 10.1373/clinchem.2017.273698. PubMed PMID: 29348166.
16. Fan J, Zhang H, Nguyen DT, Lyon CJ, Mitchell CD, Zhao Z, Graviss EA, Hu Y. Rapid diagnosis of new and relapse tuberculosis by quantification of a circulating antigen in HIV-infected adults in the Greater Houston metropolitan area. BMC medicine. 2017;15(1):188. doi: 10.1186/s12916-017-0952-z. PubMed PMID: 29089034; PubMed Central PMCID: PMC5664577.
17. Liu C, Lyon CJ, Bu Y, Deng Z, Walters E, Li Y, Zhang L, Hesseling AC, Graviss EA, Hu Y. Clinical Evaluation of a Blood Assay to Diagnose Paucibacillary Tuberculosis Via Bacterial Antigens. Clinical chemistry. 2018. doi: 10.1373/clinchem.2017.273698. PubMed PMID: 29348166.