Sustained elevated levels of C-reactive protein and ferritin in pulmonary tuberculosis patients remaining culture positive upon treatment initiation

Abstract

Background: Clinical trials that evaluate new anti-tubercular drugs and treatment regimens take years to complete due to the slow clearance of Mycobacterium tuberculosis infection and the lack of early biomarkers that predict treatment outcomes. Host Inflammation markers have been associated with tuberculosis (TB) pathogenesis. In the present study, we tested if circulating levels of C-reactive protein (CRP) and ferritin reflect mycobacterial loads and inflammation in pulmonary TB (PTB) patients undergoing anti-tuberculous therapy (ATT).

Methods: Prospective measurements of CRP and ferritin, used as readouts of systemic inflammation, were performed in cryopreserved serum samples from 165 Brazilian patients with active PTB initiating ATT. Associations between levels of these laboratory parameters with mycobacterial loads in sputum as well as with sputum conversion at day 60 of ATT were tested.

Results: Circulating levels of both ferritin and CRP gradually decreased over time on ATT. At pre-treatment, concentrations of these parameters were unable to distinguish patients with positive from those with negative acid-fast bacilli (AFB) in sputum cultures. However, patients who remained with positive cultures at day 60 of ATT exhibited heightened levels of these inflammatory markers compared to those with negative cultures at that time point.

Conclusions: CRP and Ferritin levels in serum may be useful to identify patients with positive cultures at day 60 of ATT.

Fig 1. Circulating levels of ferritin, but not CRP, substantially decrease in pulmonary TB patients undergoing anti-tuberculous therapy (ATT).

(A) Serum concentrations of ferritin and CRP were prospectively measured in a cohort of 165 treatment-naïve patients with culture confirmed pulmonary TB. Lines and whiskers represent median and interquartile values. Data were analyzed using Kruskal Wallis test with Dunn’s multiple comparisons. Statistically significant differences are highlighted. *P<0.05; **P<0.01. (B) Serum levels of Ferritin and CRP were tested for correlations at different study time points. Data were analyzed using the Spearman’s rank test. Non-linear curve fit analysis was used to illustrate the trends in data distribution in the correlation plots. (C) Concentrations of ferritin and CRP were examined prospectively in patients stratified by HIV infection status.

Fig 2. CRP and Ferritin concentrations in serum of treatment-naïve pulmonary TB patients are not significantly influenced by mycobacterial loads in sputum.

(A) CRP and ferritin levels were compared between pulmonary TB patients with negative or positive identification of acid-fast bacilli (AFB) in sputum smears using the Mann-Whitney test (CRP: p = 0.094, Ferritin: p = 0.830). (B) These markers were further compared between groups of TB patients presenting with different sputum smear grades (**p<0.01, AFB ≥3+ vs. 0) or (C) those exhibiting different culture grades using Kruskal Wallis test with Dunn’s multiple comparisons. Statistically significant differences are highlighted.

Fig 3. Pulmonary TB patients remaining with culture positive after 60 days of ATT displayed heightened levels of serum ferritin than those who had negative cultures.

(A) Changes in hemoglobin levels following initiation of ATT are shown. (B) Hemoglobin levels upon ATT initiation in TB patients whose sputum cultures became negative at day 60 of ATT and in those who remained culture positive. (C) Serum concentrations of CRP and ferritin were compared between the indicated timepoints of ATT in the group of TB patients whose sputum cultures became negative at day 60 of ATT and in that of patients who remained culture positive. In (A), (B) and (C), data were compared using Kruskal Wallis test with non-parametric linear trend ad hoc test or Dunn’s multiple comparisons (for CRP). Statistically significant differences are highlighted. (D) CRP and Ferritin levels were compared between individuals with culture negative and those with culture positive at day 60 of ATT using the Mann-Whitney U test. (E) Performance of each marker in identifying patients who had positive cultures at day 60 of ATT was tested using Receiver Operator Characteristics (ROC) curve analysis.