Abstract
Mycobacterium tuberculosis (Mtb) is extremely recalcitrant to antimicrobial chemotherapy requiring 6 months to treat drug-sensitive tuberculosis (TB). Despite this, 4–10% of cured patients will develop recurrent disease within 12 months after completing therapy. Reasons for relapse in cured TB patients remains speculative, attributed to both pathogen and host factors. Populations of dormant bacilli are hypothesized to cause relapse in initially cured TB patients however, development of tests to convincingly demonstrate their presence at the end of anti-TB treatment has been challenging. Previous studies have indicated the utility of culture filtrate supplemented media (CFSM) to detect differentially culturable tubercle bacilli (DCTB). Here, we show that 3/22 of clinically cured patients retained DCTB in induced sputum and bronchoalveolar lavage fluid (BALF), with one DCTB positive patient relapsing within the first year of completing therapy. We also show a correlation of DCTB status with “unresolved” end of treatment FDG PET-CT imaging. Additionally, 19 end of treatment induced sputum samples from patients not undergoing bronchoscopy were assessed for DCTB, identifying a further relapse case with DCTB. We further show that induced sputum is a less reliable source for the DCTB assay at the end of treatment, limiting the utility of this assay in a clinical setting. We next investigated the host proteome at the site of disease (BALF) using multiplexed proteomic analysis and compared these to active TB cases to identify host-specific factors indicative of cure. Distinct signatures stratified active from cured TB patients into distinct groups, with a DCTB positive, subsequently relapsing, end of treatment patient showing a proteomic signature closer to active TB disease than cure. This exploratory study offers evidence of live Mtb, undetectable with conventional culture methods, at the end of clinically successful treatment and putative host protein biomarkers of active disease and cure. These findings have implications for the assessment of true sterilizing cure in TB patients and opens new avenues for targeted approaches to monitor treatment response.
Highlights
Mycobacterium tuberculosis (Mtb) is a highly complex and welladapted pathogen that causes tuberculosis (TB)
There were 22 clinically cured patients analyzed for FDG Positron Emission Tomography (PET)-CT activity recruited for assessment of differentially culturable tubercle bacilli (DCTB) in sputum and bronchoalveolar lavage fluid (BALF) (Table 1)
In addition to the set of 22 patients with sputum and BALF samples, a further 19 clinically cured patients were recruited for assessment of DCTB in their induced sputum only, identifying a further two DCTB positive patients with one of these subsequently relapsing within a year of completing therapy (Table 2)
Summary
Mycobacterium tuberculosis (Mtb) is a highly complex and welladapted pathogen that causes tuberculosis (TB). Mtb mRNA can be detected in sputum and bronchoalveolar lavage fluid (BALF) in a significant fraction of cured patients at the end of curative treatment, possibly indicating residual live Mtb (Malherbe et al, 2016) It is well-established that Mtb, amongst other bacteria, can form physiologically heterogeneous populations both in-vitro and in-vivo (Balaban et al, 2004; Gefen and Balaban, 2009; Lewis, 2010; Manina et al, 2015; Walter et al, 2015; Fisher et al, 2017). A sub-population of metabolically distinct bacilli, defined as differentially culturable tubercle bacilli (DCTB) can be detected in sputa when grown in the presence of sterilized culture filtrate supplemented media (CFSM) This sub-population appears to become more prominent during early chemotherapy (Mukamolova et al, 2010; Chengalroyen et al, 2016), yet is not directly detectable by conventional microbiological diagnostic methods. Hostspecific factors are likely to be critical in determining favorable or unfavorable outcomes even in the presence of persistent bacteria
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