Changes in Mycobacterium tuberculosis-Specific Immunity With Influenza co-infection at Time of TB Diagnosis.

Background: Prior Influenza A viral (IAV) infection has been shown to increase susceptibility to tuberculosis (TB) and TB has also been shown to be a primary cause of death during pandemics, including the Spanish Influenza outbreak of 1918-1919. The majority of data has been obtained from mouse models, thus the aim of this study was to determine the impact of Flu co-infection on host immunity and disease severity in TB patients at diagnosis. Methods: Sputum from 282 patients with active TB were analyzed for presence of FluA/FluB RNA at presentation using multiplex PCR. Sputum RNA was also analyzed for Mycobacterium tuberculosis (Mtb) load using 16S RNA amplification. Supernatants from digested sputum and Mtb antigen-stimulated whole blood were analyzed using multiplex cytokine arrays and PBMC were analyzed for cytokine production from CD4+ T, CD8+ T and Mucosal Associated Invariant T cells (MAITs). Results: 12 (4.3%) of TB patients were found to have FluA or FluB viral RNA present in their sputum at the time of TB diagnosis. The TB/Flu co-infected patients had a significantly higher bacterial load compared to those with TB mono-infection (p = 0.0026). They had lower levels of IL17A in ex vivo sputum (p = 0.0275) and higher MCP-1 (CCL2) levels in the blood following PPD stimulation (p = 0.0267). TB/Flu co-infected subjects had significantly higher IFN-γ+IL-17+CD4+ and IFN-γ+IL-17-CD8+ cells compared to TB mono-infected subjects. Conclusions: These data show that Flu co-infection at time of TB diagnosis is associated with a higher bacterial load and differential cellular and soluble profiles. These findings show for the first time the impact of TB/Flu co-infection in a human cohort and support the potential benefit of Flu vaccination in TB-endemic settings

Broad adaptive immune responses to M. tuberculosis antigens precede TST conversion in tuberculosis exposed household contacts in a TB-endemic setting.

BACKGROUND: The identification of Mycobacterium-tuberculosis (Mtb) infected individuals remains a challenge due to an insufficient understanding of immune responses detected with the current diagnostic tests for latent tuberculosis i.e. the tuberculin skin test (TST) or IFN-γ release assays (IGRAs) and an inability to distinguish infection stages with current immunologic assays. Further classification based on markers other than IFN-γ may help to define markers of early Mtb infection. METHODS: We assessed the TST status of Mtb-exposed household contacts at baseline and at 6 months. Contacts were classified into those with initial positive TST (TST+); those with baseline negative TST but TST conversion at 6 months (TST converters, TSTC) and those with persistently negative TST (PTST-). We assessed their short- and long-term immune responses to PPD and ESAT-6/CFP-10 (EC) via IFN-γ ELISPOT and a multiplex cytokine array in relation to TST status and compared them to those of TB cases to identify immune profiles associated with a spectrum of infection stages. RESULTS: After 1 and 6 days stimulation with EC, 12 cytokines (IFN-γ, IL-2, IP-10, TNF-α, IL-13, IL-17, IL-10, GMCSF, MIP-1β, MCP-3, IL-2RA and IL-1A) were not different in TSTC compared to TST+ suggesting that robust adaptive Mtb-specific immune responses precede TST conversion. Stratifying contacts by baseline IFN-γ ELISPOT to EC in combination with TST results revealed that IP-10 and IL-17 were highest in the group of TST converters with positive baseline ELISPOT, suggesting they might be markers for recent infection. CONCLUSION: We describe a detailed analysis of Mtb-specific biomarker profiles in exposed household contacts in a TB endemic area that provides insights into the dynamic immune responses to Mtb infection and may help to identify biomarkers for 'at-risk' populations beyond TST and IGRA

Changes in Host Cytokine Patterns of TB Patients with Different Bacterial Loads Detected Using 16S rRNA Analysis.

Tuberculosis (TB) has overtaken HIV as the biggest infectious disease killer, with the majority of deaths occurring in sub-Saharan Africa. However it is unknown how differences in bacterial load alter host immune profiles in the sputum and blood of TB patients.16S ribosomal RNA analysis was used to determine bacterial load in sputum samples obtained from 173 patients with active TB (57 pre-treatment and 116 post-treatment). Host analyte concentrations in sputum and Mycobacterium tuberculosis (Mtb) antigen stimulated whole blood assay supernatants were analysed using multiplex cytokine arrays.Multiple logistic regression adjusting for age, sex and HIV status showed highly significant correlation of bacterial load with IL1β, IL2, IL1RA, IL4, IL6, IL8, IL9, IL15, IL17, EOTAX, FGF, IFN-γ, GCSF, MCP1, M1P1α, M1P1β, PDGF, TNFα, VEGF in sputum. With increasing time on treatment, FGF levels in sputum displayed the most significant inverse correlation with reduction in bacterial load.We show that differences in bacterial load correlates with changes in several host biomarkers. These findings have implications for development of tests for TB diagnosis and treatment response

Cytokine/chemokine responses in TB cases and household contacts stratified by TST status after stimulation with ESAT^-6/CFP^-10 (EC) at enrolment.

<p>Following overnight or 6 day culture of peripheral blood mononuclear cells with EC, supernatants were collected and multiplex cytokine assays performed. Geometric mean levels are shown (pg/ml) A: Heat map showing geometric mean levels of different analytes after overnight (D1) versus 6 day (D6) culture in cases and contacts based on TST phenotype. The highest geometric means are shaded in red and the lowest in blue. Contacts were categorized according to TST scores at baseline and 6 months: TST⁺ = TST positive: TST≥10 mm at baseline; TSTC = TST converters: TST<10 mm at baseline and TST≥10 mm plus an increase in induration of at least 6 mm by 6 month; PTST⁻: persistently TST negative: TST<10 mm at both time–points. B: Heat map showing geometric mean levels of different analytes after overnight (D1) and 6 day (D6) culture in contacts grouped according to TST status and baseline EC IFN⁻γ ELISPOT (ECS) results. The highest geometric means are shaded in red and the lowest in blue.</p

Demographic, microbiologic and Mtb exposure characteristics of study participants.

<p><b>*</b> Fisher's exact test: p = 0.017 for proximity between TST⁺ and PTST⁻. ns = not significant; TST⁺ = TST positive; TSTC = TST converters; PTST⁻ = persistently TST negative; na = not assessed; IQR = interquartile range.</p><p>Demographic, microbiologic and Mtb exposure characteristics of study participants.</p

IFN–γ ELISPOT in cases and household contacts in response to stimulation with ESAT–6/CFP–10 (EC) or Purified Protein Derivative (PPD), at enrolment.

<p>Peripheral blood mononuclear cells (PBMC) from TB cases and contacts were stimulated overnight (A and B) or for 6 days (C and D) with EC (A and C) or PPD (B and D) and an IFN–γ ELISPOT was performed. Horizontal lines represent the geometric mean of the spot forming units (SFU). Contacts were categorized according to TST scores at baseline and 6 months: TST⁺ = TST positive: TST≥10 mm at baseline; TSTC = TST converters: TST<10 mm at baseline and TST≥10 mm plus an increase in induration of at least 6 mm by 6 month; PTST⁻: persistently TST negative: TST<10 mm at both time^-points. P^-values represent comparisons adjusted for household, sex and gender.</p

Mtb bacterial load pre- and post-treatment initiation.

<p>Sputum samples from patients pre and post treatment (n = 27) were analysed using the MBL assay. Line indicates median. Data were analysed using Wilcoxon matched pairs test.</p

Correlation of MBL and host cytokines in blood and sputum.

<p>Bacterial load was analysed for each sputum sample and correlated with host biomarkers (pg/ml) in the same sputum sample and also in Mtb-stimulated and unstimulated blood using a 27-plex cytokine assay. R-values >0.4 are highlighted in yellow and significant p-values in purple. Data were analysed using a Spearman Rank Correlation. Correlation of Time-on treatment with host biomarkers in sputum was also performed.</p

Heatmap of cytokine profiles in patients with low and high bacterial loads.

<p>Cytokine levels (pg/ml) for unstimulated (NIL) and sputum supernatants were analysed using a 27-plex cytokine array. Subjects were grouped into those with low bacterial load (below the median of 48059 copies/ml) and high bacterial load (above median). Data were analysed using a Mann-Whitney U-test. NS = not significant. Colour indicates low (blue) to high (red) cytokine levels.</p

Correlation of bacterial load with cycle threshold values.

<p>RT-PCR was performed in duplicate on RNA extracted from sputum samples of TB patients. <b>A:</b> Log₁₀ bacterial load versus Ct values of Mtb present in sputum pellets. <b>B:</b> Correlation of Ct values from GeneXpert and ¹⁶S RNA analysis. Data were analysed using Spearman rank correlation.</p