A thesis submitted to the Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Doctor of
Philosophy
Johannesburg, March 2018.During tuberculosis (TB) disease, host-derived stresses and chemotherapy are thought to drive
tubercle bacilli into differential growth states. This is evidenced by the presence of
differentially culturable tubercle bacilli (DCTB) in the sputum of treatment naïve TB patients.
These bacteria do not form colonies on solid media but can be cultured following
supplementation of liquid media with culture filtrate as a source of growth stimulatory
molecules. As DCTB are non-replicating and phenotypically drug tolerant, these organisms
are thought to underpin the lengthy culture diagnosis and protracted treatment period required
for TB disease. The purpose of this study was to investigate the use of culture filtrate in
unmasking DCTB populations to: (1) quantify these populations in treatment naïve individuals,
(2) assess the response of DCTB versus conventionally culturable bacteria to first-line
treatment, (3) determine the relationship between DCTB cultured in the most probable number
(MPN) assay with other TB culture methods and (4) to enhance currently employed culture
diagnostic methods. The results from this study confirmed that treatment naïve individuals coinfected
with HIV had significantly lower quanta of DCTB in their sputum compared to their
HIV-negative counterparts. These findings implicate the host immune response in influencing
the prevalence of DCTB in sputum. During treatment, four patterns of decline in DCTB were
described. One quarter of the patient population accumulated DCTB during the first seven days
of treatment, whilst approximately the same number of individuals displayed a rapid decline in
DCTB during this period. The remaining individuals either displayed static or atypical patterns
of DCTB over the first 14 days of treatment. Following treatment completion, residual DCTB
was cultured in approximately two thirds of the patients analysed, suggesting that
bacteriological sterilization of lungs was not achieved. These observations were confirmed
using a novel fluorogenic probe specific for the detection of live Mycobacterium tuberculosis.
DCTB cultured in the MPN assay was shown to directly correlate with current TB culture
methods. These findings demonstrate a potential utility for the MPN assay in early bactericidal
activity studies to assess the sterilising effect of new TB drugs on DCTB populations.
Furthermore, the addition of culture filtrate to the BACTEC MGIT 960 assay reduced the rates
of TB detection in smear-negative, HIV-positive individuals. Collectively, these observations
demonstrate that the detection of DCTB in sputum can serve as a possible biomarker for
treatment response. Further long term studies are required to determine if DCTB can use used
to assess the risk of relapse disease and to test the efficacy of new drugs on persistent bacterial
populations.LG201
