Assessment of tuberculosis disease activity in people infected with Mycobacterium tuberculosis and living with HIV: A longitudinal cohort study

Abstract

Background: Early detection of asymptomatic incipient tuberculosis (TB) could improve clinical outcomes and reduce the spread of Mycobacterium tuberculosis (MTB) infection, particularly in HIV endemic settings. This study assessed TB disease activity over 5 years in people living with HIV co-infected with MTB using a surrogate biomarker.

Methods: Between Jan 1, 2013 and Aug 31, 2018, 2014 people living with HIV were screened annually for active TB using the Xpert MTB/RIF diagnostic assay in 11 clinics in Kenya, Tanzania, Uganda, and Nigeria. Longitudinal blood mononuclear cell samples from 46 selected patients with active and recurrent tuberculosis, latent infection, or incipient TB were further analysed for MTB-specific T-cell activation (defined by CD38 expression) as a well-defined surrogate marker for TB disease covering a total of 1758 person-months.

Findings: MTB-specific CD4 T-cell activation differentiated active, Xpert MTB/RIF positive TB from latent TB with a sensitivity and specificity of 86% and was reduced upon TB treatment initiation. Activated MTB-specific T cells were present in 63% and 23% of incipient TB cases 6 and 12 months before diagnosis of active disease, respectively. Transient increases of MTB-specific T cell activation were also observed in individuals with latent infection, while persistent activation was a hallmark of recurrent TB after the end of treatment.

Interpretation: In most cases, progression to active TB disease started 6-12 months before diagnosis by clinical symptoms and sputum occurrence of bacilli. Blood biomarkers could facilitate early detection of incipient TB, improve clinical outcomes, and reduce the transmission of MTB.

Funding: This work was supported by the President's Emergency Plan for AIDS Relief via a cooperative agreement between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense [W81XWH-11-2-0174, W81XWH-18-2-0040] and by the Bundesministerium für Bildung und Forschung (BmBF) through funding of the Deutsches Zentrum für Infektionsforschung (DZIF, TTU-TB personalized medicine TTU 02_813).

Keywords: Biomarker; HIV; Incipient tuberculosis; Tuberculosis.

Figure 1

Flowchart describing the selection of active TB cases, incipient TB cases and controls with latent TB included for analyses. Cases of active TB were defined by a positive Xpert MTB/RIF test result at the study baseline and initiation of TB therapy. Incipient TB cases were identified based on absence of TB symptoms and a negative Xpert MTB/RIF test result at baseline with subsequent development of Xpert MTB/RIF-confirmed aTB and initiation of TB treatment. LTBI was defined by a detectable IFNγ response after in vitro stimulation with MTB antigens, continuous Xpert MTB/RIF negativity, no known history of aTB and absence of aTB symptoms throughout the study period. The number of participants, visits and total person months covered by the analyses is indicated for each group. Selection of participants to be included was based on completeness of available PBMC sample set. More details on the selection of PBMC samples for analyses are also provided in the supplementary material. Abbrevations: MTB=Mycobacterium tuberculosis, TB=tuberculosis, aTB=active tuberculosis, LTBI=latent tuberculosis infection, IFNγ=interferon gamma.

Figure 2

Exemplary longitudinal phenotypic changes on MTB-specific CD4 T cells representative for PEOPLE LIVING WITH HIV with active TB, incipient TB and LTBI. Flow cytometry dot plots for IFNγ and CD38 staining on CD4 T cells are shown for four different time points. IFNγ+ MTB-specific CD4 T cells are highlighted with a red gate. The SPICE pie chart slices show 16 different IFNγ+ MTB-specific cell subsets as defined by expression of Ki67 (purple archs) HLA-DR (pink archs), CD38 (red archs) and CD27 (green archs). The 16 individual pie chart slices therefore visualize all possible combinatorial expression patterns for the tested markers Ki67, CD38, HLA-DR and CD27. The green slices visualize the proportion of MTB-specific CD4 T cells expressing CD27, but no CD38, HLA-DR or Ki67. Red slices indicate expression of 2 or 3 activation markers without expression of CD27. All other marker combinations are orange. The time point indicated is provided in relation to the time point of diagnosis and treatment initiation for cases of active and incipient TB. The Xpert MTB/RIF test and TAM-TB assay results are indicated for each time point. MTB-specific T cells were analyzed after in vitro re-stimulation of PBMC with Purified Protein Derivative. NA: not applicable. Abbrevations: MTB=Mycobacterium tuberculosis, TB=tuberculosis, aTB=active tuberculosis, LTBI=latent tuberculosis infection, IFNγ=interferon gamma, HLA-DR=Human Leucocyte Antigen-DR, TAM-TB assay=T cell activation marker-Tuberculosis assay.

Figure 3

Frequencies of CD38+ MTB-specific IFNγ+ CD4 T cells differentiate time points of patients with active TB disease (before starting versus after treatment) and LTBI. The figure shows (A) the frequency of MTB-specific CD4 T cells that express CD38 (as %IFNγ+ cells) at time of aTB diagnosis (red dots, n=21), as compared to their frequencies after TB treatment (green squares, n = 20). The MTB-specific cell activation was measured by CD38 expression analyses on MTB-specific IFNγ+ cells and shows a significant decline after TB treatment, whereas the CD38 expression on total CD4+ T cells (B) was not influenced by TB treatment. The frequency of CD38+ MTB-specific CD4 T cells (as %IFNγ+ cells) is compared between participants with aTB and LTBI in (C). Color shading indicates the range of CD38+ cell frequencies (green<20.25%, yellow 20.25% to 27.25%, orange 27.25% to 35.20%, red >35.20%); The median and interquartile ranges are indicated. The separation of the distribution for each group was tested using the Mann-Whitney test. ROC analysis (D) was used to determine 20.25% CD38+ cells (% of IFNγ+ cells) as the optimal cut-off to differentiate between patients with aTB and the LTBI/healthy group with a sensitivity of 86% and “specificity” of 86%. A frequency of IFNγ+ cells above 35.20%, which corresponds to the highest frequency of CD38+ MTB-specific CD4 T cells detected in the LTBI/healthy group, defined patients with TB with 100% specificity. CD38 frequencies from 20.25% to 27.25% and from 27.25% to 35.20% were further delineated in this analysis. These cut-offs are the basis for the heat map in Figure 4. Abbrevations: MTB=Mycobacterium tuberculosis, TB=tuberculosis, aTB=active tuberculosis, LTBI=latent tuberculosis infection, IFNγ=interferon gamma

Figure 4

Frequencies of CD38+ MTB-specific IFNγ+ CD4 T cells over time in AFRICOS study participants with LTBI, incipient TB and active TB. The figure shows the frequencies of MTB-specific CD4 T cells that express CD38 (as % IFNg+ cells) over time using the indicated color code. (A) shows CD38+ cell frequencies from participants with active and incipient TB from time points before and after TB diagnosis (X axis, study months). A positive Xpert MTB/RIF test result is indicated by an “X+”, black lines show the initiation and duration of HZRE TB treatment for each patient with TB. A blue line indicates initiation and duration of INH preventive treatment. CD38+ cell frequencies from participants with LTBI are shown in (B) together with their ART, baseline QuantiFERON (QFT) data. (NR: no response). TB=tuberculosis. Abbrevations: MTB=Mycobacterium tuberculosis, TB=tuberculosis, aTB=active tuberculosis, LTBI=latent tuberculosis infection, IFNγ=interferon gamma, HZRE=isoniazid, pyrazinamide, rifampicin and ethambutol, INH=Isonazid.