Development of a point-of-care colour reaction-based multiplex diagnostic technology for detection of HIV and TB co-infection

Currently, there are point-of-care (POC) tests for the detection of HIV and Mycobacterium tuberculosis ( M.tb ), however none can detect these pathogens simultaneously. We developed a colorimetric multiplex HIV and active TB diagnostic technology, using HIV-1 p24 and M.tb CFP10, ESAT6 and pstS1 antigens immobilized in high-binding 96 well plates or on epoxy-coated microarray slides. These antigens were used to bind corresponding antibodies in human serum at concentrations commonly found in infected individuals. This was followed by the addition of HRP conjugated secondary antibodies using either 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) or 3,3',5,5'-tetramethyl Benzidine (TMB) substrates. The sensitivity of the assay in the plates was compared to epoxy-coated slides and the latter had a better limit of detection, averaging 815-fold lower than the plates. Also, the TMB substrate produced better sensitivity than ABTS. Furthermore, we studied the specificity of the antigen-antibody reactions for this technology and determined that it was highly specific. Lastly, we investigated the stability of our technology at different pH, temperature, and storage conditions and found that it was optimal at pH 7.4, 25 ˚C, and dry storage for 1 to 90 days. Thus, the developed diagnostic technology has potential for the detection of HIV and TB co-infection at POC.