Development and assessment of a novel magnetic nanoparticle antibody-conjugate and aptamer-based assay (MNp-Ab-Ap assay) for the rapid diagnosis of pleural tuberculosis

Background: Pleural tuberculosis (pTB) is a diagnostic challenge because of its non-specific clinical features, lack of accurate diagnostic tools and paucibacillary nature of the disease.Methods: We, here describe the development of a novel magnetic nanoparticle antibody-conjugate and aptamer-based assay (MNp-Ab-Ap assay) targeting 4 different Mycobacterium tuberculosis (M.tb.) antigens (GlcB, MPT51, MPT64 and CFP-10) for pTB diagnosis.The MNp-Ab-Ap assay was developed by conjugating polyclonal antibodies on the surface of magnetic nanoparticles (MNPs) by using EDC-NHS chemistry.These conjugated MNPs were used to capture M. tb.antigens present in the pleural fluid samples.The resulting antigen-antibody complex was detected by antigen-specific 5'-biotinylated aptamers.All assays were standardized using samples of the 'Development set' (n=17) and evaluated in the 'Validation set' (n=114) in a blinded manner.Patient categorization was done using a 'Composite Reference Standard'.Assay cut-offs were determined from the 'Development set' (n=17; 'Definite & Probable' pTB; n=9 and 'Non-TB'; n=8) by calculating mean+3SD of OD450 values of the 'Non-TB' group and applied to 'Validation set ' (n=114; 'Definite' pTB; n=8, 'Probable' pTB; n=34, 'Possible' pTB; n=44).Results: Out of the 4 assays, MPT51-based MNp-Ab-Ap assay performed the best with 66.6% (95%CI;50.4-80.4)sensitivity and 95.4% (95%CI;85.1-99.4)specificity in the combined 'Definite and Probable' pTB group.Xpert MTB/RIF assay detected only six samples in the 'Validation set'.Binary logistic regression analysis indicated that MPT51-based MNp-Ab-Ap assay provided an incremental advantage over the existing diagnostic algorithm for pTB.Conclusions: We conclude that MPT51-based MNp-Ab-Ap assay is a novel technique that can pave the way towards rapid and accurate diagnosis of pTB.