BACKGROUND: Despite the enormous global burden of tuberculosis (TB), conventional approaches to diagnosis continue to
rely on tests that have major drawbacks. The improvement of TB diagnostics relies, not only on good biomarkers, but also
upon accurate detection methodologies. The 10-kDa culture filtrate protein (CFP-10) and the 6-kDa early secreted antigen
target (ESAT-6) are potent T-cell antigens that are recognised by over 70% of TB patients. Aptamers, a novel sensitive and
specific class of detection molecules, has hitherto, not been raised to these relatively TB-specific antigens.
METHODS: DNA aptamers that bind to the CFP-10.ESAT-6 heterodimer were isolated. To assess their affinity and specificity to
the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer
was evaluated by ELONA using sputum samples obtained from 20 TB patients and 48 control patients (those with latent TB
infection, symptomatic non TB patients, and healthy laboratory volunteers). Culture positivity for Mycobacterium tuberculosis
(Mtb) served as the reference standard. Accuracy and cut-points were evaluated using ROC curve analysis.
RESULTS: Twenty-four out of the 66 aptamers that were isolated bound significantly (p,0.05) to the CFP-10.ESAT-6
heterodimer and six were further evaluated. Their dissociation constant (KD) values were in the nanomolar range. One
aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and
68.75% using Youden’s index and 35% and 95%, respectively, using a rule-in cut-point.
CONCLUSION: This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6
aptamers applied to human sputum samples is feasible.
We thank Megan Lucas (Colorado State University) for providing us with
the CFP-10 and ESAT-6 expression plasmids, as well as the anti-CFP-10
antibody. We thank Lionel Gresh for his help with the ELONA
optimisation, Marisa Joubert and Stoyan Stochev for their help with the
Conceived and designed the experiments: LSR CM KD JT MK.
Performed the experiments: LSR CM. Analyzed the data: LSR CM KD
JT MK. Contributed reagents/materials/analysis tools: KD. Wrote the
paper: LSR CM KD JT MK.