In 2019, before the arrival of coronavirus, tuberculosis (TB) was the topmost cause of death worldwide from a particular infectious agent. In fact, the mortality from TB has increased in 2020 due to the current pandemic affecting essential TB services and consequently, the years of global progress in ending the TB epidemic.

TB is caused by a rod-shaped bacterium Mycobacterium tuberculosis. The most common type of TB is pulmonary tuberculosis, which affects the lungs. If the infection is not contained by the immune system, it can spread to other organs resulting in extrapulmonary TB (EPTB).

“An accurate and timely diagnosis of EPTB remains a challenge that motivated us to develop molecular probes to detect EPTB,” says Avinash Bajaj, Professor at the Regional Centre for Biotechnology (RCB), Haryana. 

Led by Bajaj, a collaborative research work was carried out by a group of researchers from RCB, Haryana; Indian Institute of Technology Mandi, Himachal Pradesh; National Institute of Immunology, New Delhi; and All India Institute of Medical Sciences, New Delhi. They report on the development of a fluorescent probe to detect mycobacteria from human tissue samples. 

Pulmonary TB is detected by analysing sputum samples. However, detecting EPTB is difficult as it is done on human tissue samples obtained by biopsy; these tissue sections often contain a very low number of mycobacteria. This count is further reduced due to the chemical processing (fixation) necessary to preserve the tissues. The existing diagnostic tests are not accurate to diagnose EPTB. Fluorescent probe-based newer diagnostic methods are more sensitive but these require specific enzyme activity of mycobacteria. This poses a problem in tissues where these enzymes lose activity due to fixation.

Recently, when antibiotic resistance has become a mounting concern, antimicrobial peptides (AMPs) have drawn attention as novel therapeutic agents. Produced by our immune system, AMPs target the cell membrane of invading microorganisms. A specific peptide – lipid complex is formed in this event, eventually killing the invader. One of the naturally-occurring bile acids that has structural similarity with AMPs is cholic acid (CA), and modified CA derivatives that mimic AMPs show broad-spectrum antimicrobial activity.

The mycobacterial cell membrane consists of distinctive lipids, and is a potential target. Thus, Bajaj’s team hypothesized that fluorophore derivatives of CA with suitable modifications could specifically bind those unique membrane lipids, thereby allowing accurate detection. They synthesized four probes of different molecular structures with varying binding capacities and tested them against different mycobacterial strains. After selecting the most effective probe (P4), they showed that it selectively binds mycobacteria over other bacteria in a polymicrobial culture as well as in mice or human tissue sections following fixation. Bajaj mentions that, ​“This probe can detect one mycobacterium in the presence of 10,000 other bacteria, depicting its sensitivity.”

In the near future, the team plans to modify these probes by coupling them to magnetic resonance imaging agents, so that these probes can also be used as a non-invasive method to detect mycobacteria. An inflammatory bowel disease that closely resembles GI-TB (gastrointestinal TB) is Crohn’s disease; it confounds diagnosis and treatment. Hence, the team will also work on differentiating GI-TB patients from Crohn’s disease patients, and correlate its findings with clinical diagnosis, says Bajaj. 

Commenting on this work, Raghunand R. Tirumalai, Principal Scientist at the Centre for Cellular and Molecular Biology, Hyderabad, who studies Mycobacterium tuberculosis, says, ​“EPTB represents an estimated one-fifth of all notified cases. Hence, this is a potential game changer for the accurate diagnosis and subsequent treatment of this class of infections. Although setting up fluorescence microscopy-based TB diagnostic facilities would be a challenge in resource-poor settings, the investment would be more than offset by the clinical benefits that will accrue from this new detection approach.”