Browsing by Subject "Mycobacterium"

Now showing 1 - 3 of 3
  • No Thumbnail Available
    Item
    Open Access
    A description of the pharmacokinetics of first line anti-tuberculosis drugs and adjunctive corticosteroid therapy in the pericardial compartment and their effect on cytokine profiles in the pericardial compartment of patients with tuberculous pericarditis
    (2025) Shenje, Justin Tapiwa; Ross, Ian
    Tuberculous pericarditis is caused by Mycobacterium tuberculosis infection of the pericardium, which is associated with a higher mortality, compared with pulmonary tuberculosis. Constrictive pericarditis, a significant complication of tuberculous pericarditis, contributes to this elevated mortality. Adjunctive corticosteroid therapy with prednisolone, has been shown to reduce the incidence of constrictive pericarditis by more than 50%. However, this is associated with significant side effects, especially in patients with HIV co-infection. Constrictive pericarditis is only partially responsible for this increased mortality, whereas other factors including delayed diagnosis of tuberculosis, cardiac complications for example, pericardial tamponade and poor pericardial exposure of anti-TB drugs also likely contribute. The pharmacokinetics of anti-tuberculous (TB) drugs within the pericardial space remain poorly understood and until now have relied on studies of pulmonary tuberculosis. While adjunctive corticosteroids have been shown to be beneficial, there are no available data on the pharmacokinetics of prednisolone in the pericardium, and uncertainty remains as to whether an empiric dose of 120 mg prednisolone is the optimal dose for reducing the incidence of constrictive pericarditis or whether it is detectable at the site of infection, to exert its pharmacodynamic effect. Combining anti-TB treatment and high doses of prednisolone are likely to have a profound effect on the immune response, therapeutic effect and cortisol milieu in the pericardium. I was therefore interested in investigating the impact of this therapeutic combination, through direct sampling of the pericardial fluid in patients with tuberculous pericarditis. Methods: A total of 16 patients with newly diagnosed tuberculous pericarditis and a pericardial effusion requiring pericardial aspiration, were enrolled into the study. The patients were randomly assigned to 120 mg of prednisolone or matching placebo and were simultaneously started on first line World Health Organization (WHO) anti-tuberculosis treatment, namely, rifampicin, isoniazid, ethambutol and pyrazinamide. Intensive pharmacokinetic samples were collected at the following timepoints, just before administration of the anti-tuberculosis treatment and prednisolone or placebo and at 0.5 hour, 1 hour, 2 hour, 3 hour, 5 hour, 8 hour and 24 hour intervals thereafter for the first 24 hours. Intensive pharmacokinetic samples were collected from plasma, pericardial fluid and saliva. The pharmacokinetics of rifampicin, isoniazid, ethambutol and pyrazinamide in the pericardium were described so as to assess the adequacy of conventional doses of the WHO first line anti-TB treatment regimen. The study also described the pharmacokinetics of 120 mg of prednisolone and evaluated its effect on endogenous corticosteroids and cytokines. Furthermore, the study compared the immunological response to tuberculous pericarditis in plasma and pericardium. Results: While pericardial drug exposures to isoniazid and pyrazinamide were comparable to those of plasma, there was a reduction in rifampicin and ethambutol exposure in the pericardium, which were 20%; (p<0.0010) and 55%; (p<0.0010) of their respective plasma drug exposures. The median pericardial rifampicin concentration was 0.125 mg/L, which was lower than the reference minimum inhibitory concentration (MIC) for Mycobacterium tuberculosis isolates, which was 0.208 mg/L (p=0.0010). The pharmacokinetic profile of prednisolone in the pericardium was similar to that of the plasma, but there was a 1.60 hours delay (p=0.0320) in time to maximum concentration (Tmax) in the pericardial space. Adjunctive corticosteroid therapy administered as a single dose of 120 mg of prednisolone, was associated with 83% reduction in median plasma interleukin-6 (p=0.0360), a 50% reduction in median plasma interleukin-8 (p=0.0300) and a 50% reduction in median pericardial interleukin-8 (p=0.0400). Prior to administration of glucocorticoids, the pericardial cortisol/ cortisone ratio was twice that of plasma (p=0.0050). A similar elevation of the cortisol/ cortisone ratio at the site of infection has previously been described in the bronchial lavage fluid of patients with pulmonary tuberculosis. Among those participants who did not receive glucocorticoids, pericardial cytokine interferon-gama, tumour necrosis-alpha, interleukin-6, interleukin-8, interleukin-10, and interferon gamma-induced protein-10 concentration increases were multiples fold higher than their corresponding plasma concentrations, (p=0.0011), (p=0.0043), (p=0.0009), (p=0.0001), (p=0.0054) and (p=0.0031), respectively. Conclusion: By directly sampling the pericardial fluid among patients with tuberculous pericarditis, I was able to gain novel insights, which have never previously been described. The reduced pericardial rifampicin and ethambutol exposures, below the required minimum inhibitory concentrations in the pericardial space, are of great concern, particularly, considering the relative importance of rifampicin in the treatment of drug sensitive tuberculosis. This warrants a revision of the dosing strategy, or a review of therapeutic regimen used in tuberculous pericarditis, to enhance efficacy. Prednisolone administered at a dose of 120 mg daily orally is detectable with high exposure in the pericardium and may explain the beneficial effect observed in tuberculous pericarditis among HIV negative individuals, through its alteration in the cytokine milieu, and lower propensity to developing constrictive pericarditis. Additionally, the raised cortisol/ cortisone ratio at the site of infection, the pericardium, compared with other matrices especially plasma may indicate an immunomodulatory role either to dampen an excessive or evoke an immunological response.
  • Thumbnail Image
    Item
    Open Access
    Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics
    (2020-03-23) Kriel, Nastassja L; Heunis, Tiaan; Sampson, Samantha L; Gey van Pittius, Nico C; Williams, Monique J; Warren, Robin M
    Abstract Background Transcriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, is in part mediated by several nucleic-acid associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acid-associated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification. Conclusions Effective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA- and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase β-subunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress.
  • Thumbnail Image
    Item
    Open Access
    Sputum lipoarabinomannan (LAM) as a biomarker to determine sputum mycobacterial load: exploratory and model-based analyses of integrated data from four cohorts
    (2022-04-02) Jones, Aksana; Saini, Jay; Kriel, Belinda; Via, Laura E; Cai, Yin; Allies, Devon; Hanna, Debra; Hermann, David; Loxton, Andre G; Walzl, Gerhard; Diacon, Andreas H; Romero, Klaus; Higashiyama, Ryo; Liu, Yongge; Berg, Alexander
    Background Despite the high global disease burden of tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb) infection, novel treatments remain an urgent medical need. Development efforts continue to be hampered by the reliance on culture-based methods, which often take weeks to obtain due to the slow growth rate of Mtb. The availability of a “real-time” measure of treatment efficacy could accelerate TB drug development. Sputum lipoarabinomannan (LAM; an Mtb cell wall glycolipid) has promise as a pharmacodynamic biomarker of mycobacterial sputum load. Methods The present analysis evaluates LAM as a surrogate for Mtb burden in the sputum samples from 4 cohorts of a total of 776 participants. These include those from 2 cohorts of 558 non-TB and TB participants prior to the initiation of treatment (558 sputum samples), 1 cohort of 178 TB patients under a 14-day bactericidal activity trial with various mono- or multi-TB drug therapies, and 1 cohort of 40 TB patients with data from the first 56-day treatment of a standard 4-drug regimen. Results Regression analysis demonstrated that LAM was a predictor of colony-forming unit (CFU)/mL values obtained from the 14-day treatment cohort, with well-estimated model parameters (relative standard error ≤ 22.2%). Moreover, no changes in the relationship between LAM and CFU/mL were observed across the different treatments, suggesting that sputum LAM can be used to reasonably estimate the CFU/mL in the presence of treatment. The integrated analysis showed that sputum LAM also appears to be as good a predictor of time to Mycobacteria Growth Incubator Tube (MGIT) positivity as CFU/mL. As a binary readout, sputum LAM positivity is a strong predictor of solid media or MGIT culture positivity with an area-under-the-curve value of 0.979 and 0.976, respectively, from receiver-operator curve analysis. Conclusions Our results indicate that sputum LAM performs as a pharmacodynamic biomarker for rapid measurement of Mtb burden in sputum, and thereby may enable more efficient early phase clinical trial designs (e.g., adaptive designs) to compare candidate anti-TB regimens and streamline dose selection for use in pivotal trials. Trial registration NexGen EBA study (NCT02371681)