Abstract:
Tuberculosis (TB) is one of the biggest killers among infectious diseases, despite the worldwide
use of a live attenuated vaccine and several antibiotics. There are an estimated eight million new
cases per year and two million deaths annually, which are compounded by the emergence of
drug resistance TB and co-infections with HIV. As of 2004, it was estimated that more than 4% of
the world’s infected people living with active TB are in South Africa. During this period, South
Africa accounted for about 2% of the world’s new TB cases; and approximately 3% of the total
deaths due to TB.
Despite the enormous burden of TB, conventional approaches to diagnosis used today continue
to rely on tests that have major drawbacks. Many of these tests are slow and lack both sensitivity
and specificity or require expensive equipment and trained personnel. For example, sputum
smear microscopy is insensitive; the culture method is technically complex and slow; chest
radiography is non-specific; the tuberculin skin test is imprecise and the results are non-specific;
nucleic acid amplification tests and phage display are rapid but specificity and sensitivity are low;
and the GeneXpert™ addresses the problems of time and sensitivity but the machine required is
extremely expensive. Clearly, TB diagnostics need to be improved and a more specific
diagnostic assay should preferably be based on antigens that are present exclusively in
Mycobacterium tuberculosis (Mtb) and not in Bacillus Calmette-Guérin (BCG).
To help improve TB diagnostics, a systematic evolution of ligands by exponential enrichment
(SELEX) process was used to select aptamers that can specifically bind to the CFP-10/ESAT-6
heterodimer, which is an early marker of TB. Anti-CFP-10/ESAT-6 aptamers were screened
using an enzyme-linked oligonucleotide assay (ELONA) for binding affinity and specificity. A total
of 24 aptamers had significant binding to the CFP-10/ESAT-6 heterodimer. One aptamer, named
EA10, which bound to the heterodimer with a dissociation constant (KD) of 8 nM, as determined
by surface plasmon resonance (SPR), was used for a proof-of-principle in a TB diagnostic test.
Evaluation of 80 clinical sputum samples from TB patients revealed that aptamer EA10 has a
specificity of 68% and a sensitivity of 80-100%. These data bode well for the development of the
aptamer for accurate diagnosis of TB.
