Differentiation of Mycobacterium bovis and Mycobacterium tuberculosis using a novel magnetic based immunoassay

Objective:
Mycobacterium tuberculosis is the primary cause of human tuberculosis (HTB). However, evidence suggests that the burden of TB caused by Mycobacterium bovis, the agent of bovine tuberculosis, is underestimated as a cause of zoonotic TB (zTB) in humans. It is most commonly transmitted to humans through contact with infected animals and consumption of contaminated food products, and results in both pulmonary and extra-pulmonary TB. zTB is indistinguishable clinically and pathologically from TB caused by M. tuberculosis, and with 99.95% similarity at the genome level and identical 16S rRNA sequences, differentiation of these species is difficult and requires expensive molecular techniques. Differentiation is critical as M. bovis is intrinsically resistant to pyrazinamide, a front line anti-TB drug. Here we describe the development of a novel magnetic based immunoassay with higher sensitivity than ELISA which is capable of differentiating between M. bovis and M. tuberculosis.

Key results/ findings:
Gamma irradiated whole cells of M. tuberculosis H37Rv and M. bovis AF2122/97, ethanol-extracted surface antigens of these cells, and identified secreted antigens were used as targets for binder production. Selected binders, when combined in precise combinations, were found by ELISA to be capable of differentiating between M. bovis and M. tuberculosis at concentrations of 104 CFU/ mL, and no cross-reactivity with the other Mycobacterium spp. tested. To increase the sensitivity of the assay, the binders were conjugated to paramagnetic gold nanoparticles (AuFeNPs). AuFeNPs demonstrate greater physicochemical stabilities and enhanced catalytic efficiencies when compared with the conventional HRP enzyme. The magnetic and catalytic properties of these functionalised particles were found to be unaffected by bioconjugation and the developed assay was capable of simultaneous sample concentration, purification, immunological sensing and differentiation of M. bovis and M. tuberculosis at concentration ranges of 103 CFU/ mL.

Significance/Impact of study:
The WHO End TB strategy aims to eradicate HTB by 2035. The absence of M. bovis identification can compromise the efficacy of the treatment regime and influence the final outcome of the patient. Differential diagnosis needs to be an integral part of control plans to effectively direct chemotherapy, prevent antimicrobial resistance developing, facilitate the study of transmission of mycobacteria between humans and animals, and provide accurate estimations of the scale of zTB. Thus, development of low-cost, highly sensitive and specific immunological diagnostics will facilitate more rapid, accurate diagnosis of ZTB particularly in high TB burden countries.