Performance of microarray in determining percent abundance of serotypes in spiked and field samples.

<p>The percent relative abundance reported by method 4 (culture microarray) compared with the inocula for 174 serotypeable pneumococci within 70 spiked samples with multiple serotypes (filled circles) and compared with results obtained by conventional serotyping according to the reference method for 61 serotypeable pneumococci within 27 field samples with multiple serotypes (open circles). For the spiked samples, the correlation of relative abundance results between the inocula and microarray was significant (<i>p <</i> 0.001): Spearman’s <i>r</i> = 0.863 (95% CI: 0.818, 0.897). Similarly, the correlation between actual relative abundance and microarray results was significant for the field samples (<i>p <</i> 0.001): Spearman’s <i>r</i> = 0.907 (95% CI: 0.847, 0.944).</p

Isolates used to create spiked samples.

<p>¹Isolates were kindly provided by Prof. Samir Saha (Bangladesh), Assoc. Prof Fiona Russell (Fiji), Dr. Peter Adrian and Prof. Shabir Madhi (South Africa), and Prof. Kate O’Brien (United States).</p><p>²Site of isolation not known.</p><p>Isolates used to create spiked samples.</p

Sensitivity and PPV of the five methods testing the 260 field samples.

<p>The point estimates and 95% CIs for sensitivity (A) and PPV (B) are depicted. The sensitivity of method 4 is higher than those of the other methods.</p

Spiked sample testing results.

<p>For each method (labelled m1–m22), the sensitivity of detection of the major serotypes (<i>x-</i>axis) and minor serotypes (<i>y-</i>axis) is plotted on the graph, with the PPV shown in colour according to the colour bar on the right. Methods that directly tested the sample or included a culture amplification step are represented by triangles and circles, respectively.</p

Serotype distribution in field samples.

<p>A total of 307 serotypeable pneumococci (representing 49 serotypes) were identified in 260 nasopharyngeal swab samples collected from children in six countries. The 26 most common serotypes are shown here, with the remaining 23 serotypes identified combined as “other”.</p

Relationship between pneumococcal carriage densities and IL-17A levels (A) Pneumococcal nasopharyngeal (NP) carriage densities in all 65 children from Fiji was measured by qPCR.

<p>IL-17A was measured by multiplex bead array. One child was removed from the analysis as an outlier (Carriage density = 1.65x10⁵ CFU/ml, IL-17A = 1710pg/ml). The correlation with the outlier was R = -0.19, p = 0.126. Correlation between IL-17A and children with low pneumococcal carriage density (B; N = 27) or children with high pneumococcal carriage density (C; N = 27). The Spearman test was used to correlate children with pneumococcal carriage density and IL-17A levels.</p

Cytokine profiles in PBMCs from children with low and high pneumococcal carriage densities.

<p>(A) Relationship between Th17 and Tregs under normal immune homeostasis and under different diseases contexts (Figure taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129199#pone.0129199.ref036" target="_blank">36</a>]). Th17 cells are the major source of IL-17A which plays a protective role against pneumococcal infection, fungal infection and extracellular bacteria such as <i>Staphylococcus aureus</i>. However, in autoimmune and chronic inflammatory diseases such as Multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and psoriasis, IL-17A is known to be pathogenic (Adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129199#pone.0129199.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129199#pone.0129199.ref037" target="_blank">37</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129199#pone.0129199.ref038" target="_blank">38</a>]). Comparison of (B) IL-23 and IL-22 (Th17) (C) TGF-β and IL-10 (Tregs) (D) IL-6, IFN-γ and TNF-α (pro-inflammatory cytokines) levels between children with high or low pneumococcal carriage densities. Bars represent mean ± SEM. Statistical comparisons were done using an unpaired Student’s t test.</p

IL-17A levels in children with high and low pneumococcal carriage densities.

<p>(A) Carriage density >8.21 x 10⁵ CFU/ml was defined as a ‘high’ carrier and <1.67 x 10⁵ CFU/ml was defined as a ‘low’ carrier. Scatter plots show the median <b>±</b> interquartile range for children with high carriage (N = 27) and low carriage (N = 27). Statistical comparisons were done using Mann-Whitney U test. (B) IL-17A levels in PBMC supernatants from children in Fiji with high (N = 27) or low (N = 27) pneumococcal carriage densities as well as children that did not carry pneumococcus (N = 29). Bars represent mean ± SEM. Statistical comparisons were done using an unpaired Student’s t test.</p

Th17/Treg balance is different in children with low and high pneumococcal carriage densities.

<p>The Th17/Treg axis is shown for IL-17A/IL-10 (A) and IL-17A/TGF-β (B) Bars represent mean ± SEM. Comparisons were done using an unpaired Student’s test.</p

Correlation between IL-17A and other Th17 cytokines IL-22 (A) and IL-23 (B) levels in PBMCs from children with high and low pneumococcal carriage density (N = 46).

<p>The Spearman test was used to correlate the cytokine levels.</p