M. tuberculosis exosome detection for pediatric TB diagnosis

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PROJECT SUMMARY/ABSTRACT Over one million new cases of tuberculosis (TB) and 239,000 TB-related deaths occur in children each year. Young children, especially those with HIV, are more likely to present with disseminated or extrapulmonary TB and paucibacillary disease, often missed by respiratory sampling. Non-sputum, biomarker-based diagnostic tools for rapid TB detection and treatment response in children, using easily obtained specimens are urgently needed. Exosomes are small (30-100 nm) membranous extracellular vesicles (EVs) originating from endosomal cell compartments; those secreted by M. tuberculosis (Mtb) or Mtb-infected macrophages appear to play a significant role in Mtb pathogenesis. Our collaborators have developed a rapid and sensitive nanoplasmon-enhanced scattering (nPES) assay which directly detects Mtb-exosomes (Mtb-EVs) from as little as 1 L of serum. Proof- of-concept nPES assays performed with Mtb markers LpqH (19-kDa Mtb lipoprotein) and LAM distinguished adult TB from at-risk patients and normal controls, and among pediatric TB cases (including HIV+) and controls with high sensitivity and specificity. We propose using archived specimens and clinical data from the Pediatric Urgent Start of HAART (PUSH) Study (NCT02063880) and a new proposed prospective cohort of children suspected of TB with high HIV prevalence to evaluate performance of nPES detected Mtb-EVs for pediatric TB diagnosis (Aim 1), treatment response (Aim 2), and evaluation of a point-of-care platform (Aim 3). In addition to assessing conventional diagnostic performance measures, we propose to use advanced epidemiologic methods (Bayesian latent class analysis) given the context of an imperfect reference. Additional evaluation in adult TB patients and healthy controls including household contacts (adults and children) and recently BCG-vaccinated infants without TB is proposed. We hypothesize nPES detected Mtb-EVs will 1) have similar diagnostic performance to the reference of Xpert/culture among children with confirmed TB without the need for sputum, and identify additional children missed by respiratory sample, 2) will provide a useful surrogate marker of treatment response with decline in quantitative levels during successful treatment, and 3) will maintain performance with a point-of-care platform. Using cryopreserved samples from a well-characterized cohort of children with HIV who underwent intensive TB evaluation and a prospective cohort of children suspected of TB with high HIV prevalence provides opportunity for efficient evaluation of a novel diagnostic with potential for clinical impact to improve pediatric TB diagnosis.