Investigating Non-sterilizing Cure in TB Patients at the End of Successful Anti-TB Therapy

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.

Keywords: 18F-FDG PET-CT; BALF proteome analysis; Mycobacterium tuberculosis; differentially culturable tubercle bacteria; dormant; sterilizing cure; treatment response; tuberculosis.

Figure 1

Sample processing and workflow used to investigate sterilizing cure in tuberculosis (TB) patients at the end of successful anti-TB therapy. Three sample types were analyzed for DCTB M. tuberculosis in clinically cured end of treatment patients (n = 22); an induced sputum, bronchoalveolar lavage fluid (BALF) and post-bronchoscopy (PB) sputum (n = 22). These patients were also scanned using FDG PET-CT to classify lesion activity at the end of treatment. The BALF supernatant from five patients was used for multiplexed proteomics analysis using tandem mass tag (TMT 10 plex) and compared to the proteome of active TB cases. A further 19 EOT patients were assessed for DCTB in their induced sputum, only. The DCTB assay consisted of a series of confirmatory tests once growth was detected including sub-culturing on blood agar, Mycobacterium Selectatab and Middlebrook 7H11 Agar. Colonies corresponding to typical growth of M. tuberculosis were subsequently picked and confirmed for acid fast bacilli (AFB) using ZN staining before being strain typed using spoligotyping.

Figure 2

(A) Representative chest X-rays at diagnosis (Dx), month 6 (M6) and month 18 (M18) and (B) corresponding FDG PET-CT scan at M6 for cured patients with differentially culturable Mtb. The FDG PET-CT scans show 3-dimensional anterior (top panel) and transverse (lower panels) views.

Figure 3

Unique proteome signatures exist in BALF from patients with active TB compared to patients maintaining clinical cure. (A) Principle component analysis of protein intensities obtained by mass spectrometry analysis of BALF from active TB patients at baseline (TB) and patients successfully completing 6 months anti-TB therapy (EOT). Active TB cases could be distinguished from the majority of EOT cases in principle component 1, with the exception of one EOT case. Follow up analysis of this patient revealed this was a relapse case with differentially culturable M. tuberculosis. (B) Volcano plot representing the differentially regulated proteins in active TB compared to EOT. The relapse sample was considered as a TB case for the statistical test. Heatmaps of the (C) top 15 upregulated and (D) downregulated proteins from the hypothesis testing. (E) Gene enrichment analysis (GSEA) of all significantly regulated proteins against gene ontology terms. GSEA was performed using the WebGestalt webserver (Liao et al., 2019).