Abstract
BackgroundMycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins.MethodsMultiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m/z interference. The final optimized method was tested in 40 exosome samples from TB positive patients.ResultsOur enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region.ConclusionsAn enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.
Highlights
Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease
We previously demonstrated that during cellular infection with Mtb, mycobacterial products can be incorporated into host cell exosomes [3]; the presence of Mtb proteins were confirmed in exosomes isolated from a variety of fluids from infected animal models [4, 5]
limit of detection (LOD)/limit of quantification (LOQ) determination in a pooled exosome matrix Exosomes isolated from the serum of 16 TB suspects and 20 TB culture-confirmed patients were pooled to create a matrix for the determination of the LOD and LOQ for the 41 peptides in our two multiple reaction monitoring (MRM) assays
Summary
Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Current diagnostics rely on sputum availability to confirm the presence of bacteria by microscopy, live bacilli by culture or using molecular tools to detect pathogen DNA. The exosomes of interest, that is those derived from the Mtb-infected cells, are still a minor component of the entire exosome population. Coupling this purification with a sensitive downstream detection platform is critical for the detection of mycobacterial proteins as potential biomarker candidates
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