Associations between systemic inflammation, mycobacterial loads in sputum and radiological improvement after treatment initiation in pulmonary TB patients from Brazil: a prospective cohort study

Abstract

Background: Mycobacterium tuberculosis infection is known to cause inflammation and lung tissue damage in high-risk populations. Nevertheless, direct associations between mycobacterial loads, systemic inflammation and pulmonary lesions upon treatment initiation have not been fully characterized. In the present exploratory study, we prospectively depict the immune profile, microbial clearance and evolution of radiographic lesions in a pulmonary tuberculosis (PTB) patient cohort before and 60 days after anti-tuberculous treatment (ATT) initiation.

Methods: Circulating levels of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α) and C-reactive protein (CRP), as well as values of erythrocyte sedimentation rate (ESR) were measured in cryopreserved serum samples obtained from 73 PTB patients at pre-ATT and day 60 of treatment. Changes of the immune profile over time were compared with mycobacterial loads in sputum and culture conversion at day 60 of ATT. Additional analyses tested associations between improvement of chest radiographic lesions at day 60 and pre-treatment status of inflammation and mycobacterial loads.

Results: Within the inflammatory parameters evaluated, values of CRP, IL-2, IL-4, TNF-α and ESR significantly decreased upon treatment initiation. On the converse, IL-10 levels substantially increased at day 60 of ATT, whereas concentrations of IL-6 and IFN-γ remained unchanged. Multidimensional analyses revealed that ESR, IL-2, IL-4 and CRP were the parameters with the highest power to discriminate individuals before and after treatment initiation. We further demonstrated that higher bacterial loads in sputum at pre-ATT were associated with increased systemic inflammation and higher risk for positive M. tuberculosis sputum cultures at day 60 of treatment. Furthermore, we found that pre-ATT mycobacterial loads in sputum and systemic inflammation synergistically associated with the status of radiographic lesions during treatment (Relative risk for chest X-ray improvement: 10.0, 95 % confidence interval: 2.4-40.0, P = 0.002).

Conclusions: M. tuberculosis loads in sputum are directly associated to the status of systemic inflammation and potentially impact the immune profile, culture conversion and evolution of lung lesions upon ATT initiation.

Keywords: Acid-fast bacilli; Anti-tuberculous treatment; Biomarker; C-reactive protein; Erythrocyte sedimentation rate; Inflammation; Radiographic evaluation; Tuberculosis.

Fig. 1

Prospective immune profiling of pulmonary TB patients before and 60 days after anti-mycobacterial treatment initiation. a Levels of C-reactive protein (CRP), indicated cytokines and erythrocyte sedimentation rate (ESR) were measured in serum samples from 73 patients with pulmonary TB at day 0 and 60 of anti-tuberculous treatment (ATT). Data were analyzed using Wilcoxon matched pairs test. Dotted lines represent median values detected in age- and gender-matched uninfected and healthy controls (n = 10), to serve as reference. b Network analysis was performed based on Spearman correlation matrices as described in Methods. Each connecting line represents a statistically significant correlation (p < 0.05). The strength (r value) and level of significance (p-value) for each significant correlation is shown in Additional file 1: Table S1. c A model of principal component analysis (PCA) was employed to test whether combination of the markers could cluster the patients at different time points of ATT (left panel). Vector analysis was further utilized to illustrate the influence of each marker in data distribution of the PCA model (center panel). Delta variation of the indicated markers from day 0 to day 60 of ATT initiation (right panel). Data represent median and interquartile ranges. d A second model of PCA in which all variable were included except from those with the largest delta variation from day 0 to day 60 of ATT was designed. HC = healthy controls, PC = principal component

Fig. 2

Inflammatory profile, mycobacterial loads and culture positivity at day 60 of antimicrobial treatment in PTB patients. a A hierarchical clustering analysis (Ward’s method with bootstrap) was employed to show the immunological profile of TB patients with different AFB status in smear samples at pre-ATT. Data was processed as described in Methods. In the heatmap, the squares represent values below or above the median values (log10) of a given biomarker in the entire study population (n = 73). b The same hierarchical clustering analysis was performed to compare the inflammatory profile of TB patients stratified according to the AFB status in sputum smears at pre-ATT and the study time points. c Serum levels of the indicated cytokines were compared between day 0 and day 60 of ATT in TB patients presenting with positive or negative AFB in sputum smears at study enrollment. Data were analyzed using the Wilcoxon matched pairs test. Significant p-values are shown. d Left panel shows frequency of TB patients with AFB >1+ in sputum smears within the groups of individuals stratified by culture status at day 60 post ATT initiation (positive culture at day 60, n = 13; negative culture, n = 53). Data were compared using the Fisher’s exact test. Right panel shows logistic regression analysis adjusted for age, gender and hemoglobin levels was performed to determine the association between AFB sputum smear > +1 at pre-ATT and culture positivity at 60 day upon ATT initiation. e A hierarchical clustering analysis was employed to illustrate the inflammatory profile of TB patients stratified by treatment time point and culture status at day 60 after ATT initiation

Fig. 3

Association between cavitary lung disease, systemic inflammation and radiological improvement of TB lung lesions. a Levels of C-reactive protein (CRP), indicated cytokines and erythrocyte sedimentation rate (ESR) were compared between day 0 and 60 of anti-tuberculous treatment (ATT) in TB patients with (n = 42) or without (n = 31) cavitary disease. The Wilcoxon matched pairs test was used to compare changes in the biomarker levels between the timepoints in each clinical subgroup. The Mann–Whitney test was used to compare biomarker values between cavitary vs. non-cavitary TB disease at different timepoint. Dotted lines represent median values detected in age- and gender-matched uninfected and healthy controls (n = 10), to serve as reference. * p < 0.05; ** p < 0.01; *** p < 0.001. b A hierarchical clustering analysis (Ward’s method with bootstrap) was employed to illustrate the inflammatory profile of TB patients stratified by treatment timepoint and cavitary lung disease at pre-ATT. Asterisks highlight the parameters that exhibited statistically significant differences in one or more comparison [shown in (a)]. c frequency of TB patients with or without cavitary TB exhibiting AFB+ smears at pre-ATT (left panel), positive M. tuberculosis sputum culture (center panel) and improvement of lung lesions assessed by chest X-rays at day 60 of treatment.. Data were compared using the Fisher’s exact test

Fig. 4

Association between mycobacterial loads in sputum, systemic inflammation and radiological improvement of TB lung lesions. a Frequency of individuals exhibiting radiological improvement of lung lesions assessed by chest x-ray in the groups of TB patients according to the indicated AFB status in smear samples at pre-ATT. Data was analyzed using Fisher’s exact test. b Serum levels of CRP were compared between day 0 and day 60 of ATT in TB patients stratified by improvement lung lesions assessed by chest X-ray. Data were analyzed using the Wilcoxon matched pairs test. * p < 0.05; ** p < 0.01; *** p < 0.001. c Different models of regression analysis adjusted for age, gender and hemoglobin levels were employed to determine the association between low AFB sputum smear grade (<2+), low serum levels of CRP (concentrations below the median value of the entire study population, 4.7 mg/L) at pre-ATT and radiological improvement at day 60 of ATT