More about the Deformation of Our Language

<div><p>Background</p><p>Although tuberculosis is transmitted by the airborne route, direct information on the natural output of bacilli into air by source cases is very limited. We sought to address this through sampling of expelled aerosols in face masks that were subsequently analyzed for mycobacterial contamination.</p><p>Methods</p><p>In series 1, 17 smear microscopy positive patients wore standard surgical face masks once or twice for periods between 10 minutes and 5 hours; mycobacterial contamination was detected using a bacteriophage assay. In series 2, 19 patients with suspected tuberculosis were studied in Leicester UK and 10 patients with at least one positive smear were studied in The Gambia. These subjects wore one FFP30 mask modified to contain a gelatin filter for one hour; this was subsequently analyzed by the Xpert MTB/RIF system.</p><p>Results</p><p>In series 1, the bacteriophage assay detected live mycobacteria in 11/17 patients with wearing times between 10 and 120 minutes. Variation was seen in mask positivity and the level of contamination detected in multiple samples from the same patient. Two patients had non-tuberculous mycobacterial infections. In series 2, 13/20 patients with pulmonary tuberculosis produced positive masks and 0/9 patients with extrapulmonary or non-tuberculous diagnoses were mask positive. Overall, 65% of patients with confirmed pulmonary mycobacterial infection gave positive masks and this included 3/6 patients who received diagnostic bronchoalveolar lavages.</p><p>Conclusion</p><p>Mask sampling provides a simple means of assessing mycobacterial output in non-sputum expectorant. The approach shows potential for application to the study of airborne transmission and to diagnosis.</p></div

Proportion of CD4+ or CD8+ cells positive for any of the 4 factors (CD107a, IFN-γ, IL-2 or TNF-α) following overnight antigenic stimulation.

<p>Data expressed as median [interquartile range (IQR)]-positive cells per subset.</p><p>* = p≤0.05;</p><p>** = p≤0.01,</p><p>*** =  p≤0.001 compared to TB cases pre-treatment.</p

Increased CD107a, IFN-γ and TNF-α expression in TB cases pre-treatment and reduction post-treatment.

<p>Bar indicates median of TB cases pre-treatment (n = 12, filled circles), TB cases post-treatment (n = 10, open circles) and 17 HHC (triangles). Data were analysed using a Kruskal-Wallis ANOVA followed by Dunn's post-test comparison and p-values indicated.</p

The functional profile of CD4+ and CD8+ T cells is increased in TB cases but reduced post-treatment.

<p>PBMC were stimulated overnight using ESAT-6/CFP-10 and CD4+ and CD8+ cells were analysed by flow cytometry for different combinations of IFN-γ, IL-2, TNF-α and CD107a. Responses are grouped and colour coded according to the number of factors: White  = 1 function, black  =  any 2 factors, blue  =  any 3 factors and red  =  all 4 factors. The pie charts summarise the results shown in the dot plots indicating the combination of cytokines as a percent of total responding cells for CD4+ (A) and CD8+ (B). Significant differences are indicated and were determined using a Kruskal-Wallis ANOVA followed by Dunn's post-test comparison. Pie chart segments were analysed using a permutation test and p-values indicated.</p

Representative flow cytometry profiles demonstrating the gating strategy used throughout the study.

<p><b>A)</b> PBMC were stimulated overnight and cells were analysed by flow cytometry for expression of CD107a, TNF-α, IL-2 and IFN-γ. Following lymphocyte gating, CD4+ and CD8+ T cells were analysed separately for combinations of all 4 factors. B) Representative dot plots illustrating cytokine production from CD4+ and CD8+ T cells following PHA, PPD, EC and no stimulation.</p

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<p>Tuberculosis (TB) is still a global health concern, especially in resource-poor countries such as The Gambia. Defining protective immunity to TB is challenging: its pathogenesis is complex and involves several cellular components of the immune system. Recent works in vaccine development suggest important roles of the innate immunity in natural protection to TB, including natural killer (NK) cells. NK cells mediate cellular cytotoxicity and cytokine signaling in response to Mycobacterium tuberculosis (Mtb). NK cells can display specific memory-type markers to previous antigen exposure; thus, bridging innate and adaptive immunity. However, major knowledge gaps exist on the contribution of NK cells in protection against Mtb infection or TB. We performed a cross-sectional assessment of NK cells phenotype and function in four distinct groups of individuals: TB cases pre-treatment (n = 20) and post-treatment (n = 19), and household contacts with positive (n = 9) or negative (n = 18) tuberculin skin test (TST). While NK cells frequencies were similar between all groups, significant decreases in interferon-γ expression and degranulation were observed in NK cells from TB cases pre-treatment compared to post-treatment. Conversely, CD57 expression, a marker of advanced NK cells differentiation, was significantly lower in cases post-treatment compared to pre-treatment. Finally, NKG2C, an activation and imprinted-NK memory marker, was significantly increased in TST+ (latently infected) compared to TB cases pre-treatment and TST− (uninfected) individuals. The results of this study provide valuable insights into the role of NK cells in Mtb infection and TB disease, demonstrating potential markers for distinguishing between infection states and monitoring of TB treatment response.</p

Subject information.

<p>IQR = interquartile range; MA = <i>M. africanum</i>; MTb = <i>M. Tuberculosis</i>.</p

Cytokine responses following PPD stimulation.

<p>Whole blood from TB cases (n = 36), TST+ (n = 20) and TST− (n = 19) contacts was stimulated for 7 days with PPD and the supernatant assessed by multiplex cytokine analysis. A) IFN-γ. B) TNF-α, C) IL-10, D) IL-12(p40), E) IL-13, F) IL-17 and G) IL-18. Line indicates median. Data were analysed using Tweedie distributions with p-values indicating significant differences after adjustment for age and sex. H) Percentage of responders producing IFN-γ, IL-10, TNF-α, IL-12, IL-13, IL-17, IL-18. Dark bars = cases, grey bars = TST+ contacts, white bars = TST− contacts; p-values are indicated and refer to comparison with TST− contacts.</p

Flow chart demonstrating how cytokine production following TB10.4 stimulation will improve diagnosis of active TB.

<p>At the moment, patients with suspected TB may be confirmed by analysis of chest x-ray (CXR) plus sputum culture positivity in laboratories which have these capabilities. Analysis of TNF-α, IL-12(p40) and IL-17 production following TB10.4 stimulation should enhance classification into active disease or latent infection in laboratories without culture facilities and also for patients with suspected TB but smear/culture negative results (such as HIV-positive patients or extrapulmonary TB patients). These findings should be further validated in a short-term assay and in difficult to diagnose patient groups.</p

Multivariate analysis for classification into TB cases, TST+ or TST− contacts.

<p>Multivariate analysis for classification into TB cases, TST+ or TST− contacts.</p