New strategies for assessment of the persistence of viable bacilli in latent and active tuberculosis

Background: Management of tuberculosis (TB) is hampered by inadequate methods for diagnosis. Furthermore, most current methods cannot determine whether persons with TB infection harbor viable bacilli. Although nearly one third of the world population are estimated to be infected with TB, it is likely that many persons with latent TB infection (LTBI) have spontaneously resolved infection.

Furthermore, most existing bacteriological methods cannot determine viability of detected bacteria in persons with active TB.

Aims: We hypothesize that expression of cytokines and small non-coding RNAs (sncRNA) change during treatment for LTBI, and that identification of biomarker profiles can be used to assess persistence of viable bacilli. We also hypothesize that techniques based on incorporation of the glucose molecule trehalose can be used for diagnosis of active TB, and to determine viability of bacilli in patients receiving treatment for active TB.

Work plan: Blood from persons receiving treatment for LTBI will be collected at initiation and end of treatment, and 6 months later. Levels of cytokines in TB-antigen stimulated blood will be compared longitudinally in these individuals, as well as with those in persons with untreated LTBI and in patients treated for active TB. Microarrays will be performed in a subset of participants to identify differentially expressed sncRNAs, followed by PCR testing of candidate sncRNAs in plasma.

Biomarker profiles will be analysed to identify patterns suggestive of bacterial eradication during therapy.

From patients investigated for suspected active TB, respiratory and tissue samples will be tested with trehalose incorporation techniques (using fluorescence microscopy and flow cytometry), comparing detection rates with reference methods. Separately, drug sensitivity testing based on trehalose incorporation will be investigated on TB strains with and without different patterns of phenotypically detected drug resistance.

Significance: Methods that can assess the presence of viable bacilli in persons with LTBI would help target prophylactic therapy to persons at risk of active TB, and avoid unnecessary treatment for other individuals with signs of LTBI. Rapid and robust techniques that can assess bacterial viability in active TB may offer diagnostic alternatives, and may also be considered for drug sensitivity testing.