Additional file 1 of Estimating cutoff values for diagnostic tests to achieve target specificity using extreme value theory

Additional file 1. See the online Supplementary Materials for technical appendices and additional results

Oral lymphoid tissues exhibit a greater degree of histologic change than non-lymphoid tissues.

<p>The degree of histologic change in oral lymphoid tissues (retropharyngeal lymph node and tonsil) was significantly higher than in non-lymphoid tissues (tongue, buccal mucosa, and salivary gland) (p<0.001). The degree of histologic change was significantly greater in the retropharyngeal lymph node (p<0.001), palatine tonsil (p<0.001), and tongue (P = 0.002) than in the salivary gland. A greater degree of histologic change was also observed in the retropharyngeal lymph node compared to the buccal mucosa (p<0.001) (ANOVA with Tukey test for multiple comparisons).</p

FIV RNA and DNA in oral lymphoid tissues is significantly higher than levels in non-lymphoid oral tissues.

<p><b>(A)</b> FIV <i>RNA</i> levels in oral lymphoid tissues (retropharyngeal LN and tonsil) are significantly higher than non-lymphoid oral tissues (tongue, buccal mucosa, salivary gland) (p<0.0001), suggesting that oral lymphoid tissues serve as the site of viral replication and release into saliva. <b>(B)</b> FIV <i>proviral DNA</i> levels in oral lymphoid tissues are not as high as in circulating PBMC, but are significantly greater than in non-lymphoid tissues (p<0.0001), indicating that lymphoid organs may serve as oral reservoirs of FIV latency and persistence.</p

Proposed model of oral FIV pathogenesis.

<p>Similar to the gastrointestinal tract, FIV exhibits a tropism for oral tissues, providing a pathway for circulating virus to extravasate and infect resting T-lymphocytes (rT) and dendritic cells (DC). <b>(A)</b> As documented in the intestinal mucosa, infection and depletion of resting mucosal lymphocytes in the oral mucosa may cause inflammation, damage mucosal barriers, and subsequent translocation of oral microbes; resulting in lymphocyte activation and recruitment to oral mucosal tissues. <b>(B)</b> Mucosal injury initiates a chronic cycle of immune activation and provides a renewable source for target cell infection by recruiting susceptible cells to the site of injury. Peripheral FIV-infected cells traffic to oral lymphoid tissue via lymphatics <b>(C)</b> or direct migration to tonsils <b>(D)</b> resulting in antigen presentation, T-lymphocyte activation (aT), and infection of resident leukocytes, establishing a reservoir of persistent FIV replication in latently infected T-lymphocytes (iT) in oral lymphoid tissues. <b>(E)</b> FIV infected cells are likely shed from oral mucosal sites into saliva, resulting in the presence of salivary proviral DNA. Cells within oral lymphoid tissues have enhanced FIV replicative activity, resulting in a higher ratio of FIV viral RNA to proviral DNA than noted in the peripheral circulation. <b>(F)</b> FIV RNA in saliva may be achieved by direct release of virus particles from infected cells into saliva via the intimate association of tonsillar lymphoid tissue with the oral mucosa. <b>(G)</b> While FIV specific IgG responses are detected in saliva, anti-FIV IgA antibodies are not specifically enhanced, allowing FIV virus and infected cells to persist in saliva at high levels.</p

Limited IgA mucosal antibody response during FIV infection.

<p>There is a trend for salivary IgG concentrations to be slightly increased in FIV-infected cats following infection (solid blue line/squares) (mean ±SE; p = 0.216, RM-ANOVA). Salivary IgG remained constant in naïve animals (blue dashed line/clear squares). Salivary IgA concentrations did not differ significantly between FIV-infected cats (solid green line/circles) and naïve cats (dashed line/clear circles) over time (p = 0.969), but as expected, levels of IgA were higher in saliva than IgG.</p

FIV specific IgG and IgA antibodies are detected in saliva of infected cats.

<p><b>(A)</b> Anti-SU IgG and <b>(B)</b> anti-CA IgG antibody levels in saliva of FIV infected cats (solid blue lines) were significantly elevated (mean ±SE; treatment p<0.01 and p<0.001, respectively) compared to naïve animals (dotted blue lines) and increased significantly over time compared to naïve saliva (interaction anti-CA: p<0.05; anti-SU: p<0.001; RM-ANOVA). <b>(C)</b> Anti-SU IgA antibody levels in saliva of FIV infected cats (solid green lines) were significantly elevated compared to naïve animals (dotted green lines) (treatment p<0.01), but did not increase significantly over time compared to naïve saliva (interaction p = 0.569; RM-ANOVA). <b>(D)</b> No difference in salivary anti-CA IgA antibody levels was observed between FIV-positive animals and naïve animals.</p

FIV viral RNA and proviral DNA are detected in saliva of infected cats.

<p><b>(A)</b> Mean FIV viral RNA in saliva of infected cats is greater than observed in plasma (Mean: 446,000 copies/ml saliva, 85,100 copies/ml plasma). There is a trend (p = 0.165) for salivary viral load to be higher than plasma over the course of the study (RM-ANOVA). <b>(B)</b> Proviral DNA is present in saliva of infected cats, although levels tended to be 10-fold lower than circulating PBMC (p = 0.214; RM-ANOVA). <b>(C)</b> The ratio of FIV RNA to DNA is significantly higher in saliva than circulating levels in blood over the entire study (p = 0.036) and at each time point measured (day 43, p = <0.001; day 64, p = 0.014; RM-ANOVA with multiple comparisons). <b>(D)</b> Saliva from FIV-infected cats contains infectious FIV virus, as evidenced by FIV replication in GFox (CRFK) cells and the production of FIV viral particles following inoculation of saliva (day 21 post-inoculation, positive Cutoff = Mean Naïve Abs + 3*SD).</p

FIV induces mild to moderate pathology in oral tissues.

<p><b>(A)</b> Retropharyngeal lymph node. Sham-inoculated control, HE stain, 40x. <b>(B)</b> Retropharyngeal lymph node and <b>(C)</b> palatine tonsil from FIV-infected cats exhibit moderate lymphoid hyperplasia with multifocally enlarged germinal centers and thin mantle zones, 40x. Higher magnification (insets, 200x) demonstrates tingible body macrophages (arrows). HE stain. The submucosa of the tongue <b>(D)</b> and buccal mucosa <b>(E)</b> are multifocally infiltrated by small to moderate numbers of small lymphocytes and plasma cells (arrows, 100x), as well as small numbers of scattered mast cells (arrows, inset, 200x). HE stain. <b>(F)</b> Submandibular salivary gland. Minimal numbers of small lymphocytes and plasma cells multifocally surround acini (arrowheads). HE stain, 400x.</p

Total variances within infection status groups on different days.

a<p>The total variance for all cats (last row) is 11 on each day because of normalization.</p

Pearson's coefficients of the ten strongest (absolute) pair-wise correlations between immunological responses for each day of analysis.

a<p>Pair-wise correlations that increase (bold) or decrease (italics) are highlighted.</p>b<p>The sign indicates whether a pair co-varies positively or negatively.</p>c<p>All coefficients are statistically significant (p-values substantially smaller than 0.05) using a test for association/correlation between paired sample, except for the two negative correlations of Lymph with TNFα and IL10 at day 52, (TNFα p-value  = 0.13, IL10 p-value  = 0.12).</p