Most TB cases are diagnosed by slow and somewhat non-specific microbiological methods. PCR-based GeneXpert MTB/RIF, introduced to improve speed and specificity, has poor sensitivity at low bacterial loads, cannot distinguish live and nonviable bacilli, and has reduced performance in HIV and TB co-infected patients. Serum-based detection of Mtb virulence factors offers direct evidence of TB, but current methods lack adequate sensitivity and specificity. We have developed a rapid blood-based diagnostic method independent of mycobacterial isolation to quantify the low molecular-weight Mycobacterium tuberculosis (Mtb) antigens (CFP-10 and ESAT-6). Our strategy combines energy mediating porous silicon nanodisks, functionalized with customized antibodies highly specific to Mtb antigen peptides, and high-throughput mass spectrometry (NanoDisk-MS) for dual enhancement of sensitivity and specificity.
Serum samples are subjected to microwave-assisted tryptic digestion and mixed with functionalized NanoDisks and stable isotope-labeled internal standard peptides. The antibody-conjugated nanodisks performs the recognition and enrichment of target peptides and stable isotope-labeled internal standard peptides, following with a Matrix Assisted Laser Deposition Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) analysis. A NanoDisk effect to enhance MALDI-TOF MS signal allows quantification of target peptide at low concentrations, determined by MS intensity ratio of target and isotope-labeled internal standard peptides.
We evaluated our platform with 385 adult and 720 pediatric patients and controls chosen from five highly relevant cohorts (active TB, HIV/TB co-infection, pediatric TB, latent TB, and non-TB mycobacterial infection), provided by multiple institutes worldwide. Sensitivities and specificities of adults (92.1% / 98.2%) and children (89.0% / 97.5%) were achieved in active TB identification. Absolute quantification of circulating antigens was informative in detecting treatment response four days after anti-mycobacterial initiation.
Our NanoDisk-enabled TB detection assay addresses sensitivity and speed shortcomings associated with active TB diagnosis, and meets several criteria for a WHO-mandated noninvasive TB assay. Based on our preliminary studies, we are confident that this diagnostic system will benefit the global tuberculosis control effort by improving the personalized management of TB.