Tuberculosis (TB), particularly multidrug and extensively drug-resistant TB (M/XDR-TB), poses a significant, worldwide health threat to HIV-exposed children, as progression from infection to disease can occur rapidly and unpredictably. The current gold standard for TB diagnosis and treatment monitoring in children as well as adults relies heavily on time-consuming bacterial culturing or bacterial DNA detection (e.g. GeneXpert MTB/RIF) methods. Clinical diagnosis of childhood TB is especially challenging because of the paucibacillary (few bacteria) nature of the disease and difficulties in obtaining clinically relevant specimens. Even sample collection is itself an enormous task because it often requires invasive procedures (e.g., gastric aspiration, lymph node biopsy, and lumbar puncture). To minimize the destruction and health care burdens resulting from this infectious disease in vulnerable HIV-infected children, a specific and quantitative diagnostic test for active TB is needed for early identification and prevention of TB transmission. We have recently developed a biomarkers detection assay that couples the tunable properties of silica nanopore chips (referred to as Nanotrap ) to the detection capabilities of our benchtop matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS), which capitalizes on recent advances in clinical chemistry applied to MS that enables a different biomarkers detection modality not seen to date. Our preliminary studies reveal three major findings: (1) with the Nanotrap-MS platform, two TB biomarkers (CFP-10 and ESAT-6) can be detected in circulation at an incredibly low concentration (as little as 1.0 fmol), flagging the presence of possible infection well before any conventional technique can assess bacterial load; (2) we have used the platform to differentiate 51 HIV+/TB+ children from 27 HIV+/TB- children and 18 healthy controls simply by detecting the presence of CFP-10 and ESAT-6 in patients' blood, rather than performing burdensome bacterial isolation procedures, with 100% specificity and 94% sensitivity; and (3) perhaps most strikingly, the time between sample processing to answer, or diagnosis, is only half a day rather than the typical 4-6 week period required for conventional methods! In this proposal, we aim to advance the Nanotrap-MS platform much closer to full clinical translation by: a) establishing a simple but extremely accurate protocol to quantify the amount of CFP-10 and ESAT-6 in tandem in a small blood aliquot; b) performing a clinical validation study for Nanotrap- MS, using a large sample set collected from TB-infected children who may also be HIV carriers; and c) investigating the platform's feasibility in monitoring the (early) treatment efficacy of anti-TB therapies, as well as screening for the possible emergence of M/XDR-TB. Successful execution of the proposed studies will help to diminish the likelihood of or even prevent TB transmission, and improve clinical management of TB. Furthermore, the Nanotrap-MS platform has enormous potential not just for TB disease assessment, but it also has broad applications in virtually any clinical indication that manifests in biomarker release int circulation.
The current tuberculosis diagnostic methods are based on time-consuming bacterial isolation, culture, and identification (or by detection of bacterial DNA), which are especially difficult for childhood TB diagnosis due to the paucibacillary nature of the disease and difficulties in obtaining clinically relevant specimens. In this grant application we wll implement a quantitative biomarker detection and monitoring platform that combines a sophisticated peptide-enriching nanotechnology procedure ( Nanotrap ), with the powerful capabilities of bench-top mass spectrometry (MS), without the use of complicated bacterial isolation procedures or antibodies to address the challenge in the field of active TB detection and control in HIV exposed children. Our ultimate goal for this study is to use the Nanotrap-MS for early identification of TB infection in this vulnerable patient population and to assess TB biomarker fluctuations as a function of anti-TB therapies; and we are confident that this to be a high-impact strategy that could decrease the likelihood of transmission, improve therapeutic regimens, and prevent the emergence of drug resistance TB strains.
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