An Application of Outer Membrane Protein P6-Specific Enzyme-Linked Immunosorbent Assay for Detection of <i>Haemophilus influenzae</i> in Middle Ear Fluids and Nasopharyngeal Secretions

<div><p>An enzyme-linked immunosorbent assay specific to outer membrane protein P6 (P6-ELISA) was applied for detecting <i>Haemophilus influenzae</i> in middle ear fluids (MEFs) from acute otitis media (AOM) patients and in nasopharyngeal secretions (NPSs) from acute rhinosinusitis patients. P6-ELISA had a sensitivity of 83.3% for MEFs and 71.5% for NPSs and a specificity of 85.6% for MEFs and 92.5% for NPSs, respectively. Real-time PCR exhibited significant differences in the number of <i>ompP1</i> gene copies among samples determined by P6-ELISA to be positive and negative for <i>H. influenzae</i>. However, because the P6-ELISA test has the reactivity in <i>Haemophilus</i> species include two commensals <i>H. haemolyticus</i> and <i>H. parainfluenzae</i>, it is thus a weak method in order to detect only NTHi correctly. Consequently, diagnosis using the P6-ELISA should be based on an overall evaluation, including the results of other related examinations and clinical symptoms to prevent misleading conclusions in clinical setting.</p></div

Sensitivity, specificity, positive predicting value, and negative predicting values of P6-ELISA to determine the presence of <i>H. influenzae</i> in MEFs and NPS.

<p>The numbers in a parentheses show the number of samples.</p

Sensitivity and specificity of the ODK-0901 test for NPSs depending on prior antimicrobial treatment.

<p>Sensitivity and specificity of the ODK-0901 test for NPSs depending on prior antimicrobial treatment.</p

Sensitivity and specificity of the ODK-0901 test for MEFs and NPSs.

<p>Sensitivity and specificity of the ODK-0901 test for MEFs and NPSs.</p

Sensitivity and specificity of the ODK-0901 test for MEFs depending on prior antimicrobial treatment.

<p>Sensitivity and specificity of the ODK-0901 test for MEFs depending on prior antimicrobial treatment.</p

Prediction of middle ear pathogen by nasopharyngeal test.

<p>Prediction of middle ear pathogen by nasopharyngeal test.</p

Positive and negative predicting values resulting from evaluating NPSs to determine the presence of <i>H. influenzae</i> in MEFs.

<p>Positive and negative predicting values resulting from evaluating NPSs to determine the presence of <i>H. influenzae</i> in MEFs.</p

Distribution of the number of copies of <i>pspA</i> gene in MEF and NPS depending on prior antimicrobial treatment and the ODK-0901 test.

<p>The pneumococcal <i>pspA</i> gene was quantified by real-time PCR and the distribution was expressed. Open circles are culture-negative specimens. Closed circles are culture-positive specimens. A) Middle ear fluid; B) nasopharyngeal secretion.</p

Distribution of the number of copies of the <i>H.</i><i>influenzae ompP1</i> gene in MEFs and NPSs based on the results of P6-ELISA and conventional culture.

<p>Vertical axis: the number of copies of the <i>ompP1</i> gene calculated from real-time PCR. Open circle: <i>H. influenzae</i> culture negative, closed circle: <i>H. influenzae</i> culture positive. The number of copies of the <i>H. influenzae ompP1</i> gene in ODK-0902-positive arid culture-positive populations differed significantly between ODK-0902-positive and -negative populations (<i>p</i><0.001).</p

Mature DC migration is attenuated in association with reduced expression of CCR7 by OGR1 deficiency in vitro.

<p>(A and B) BMDCs from WT or OGR1-deficient mice were pulsed with 100 μg/ml of OVA as mature DCs or its vehicle as immature DCs overnight. Migration response to the indicated concentrations of ATP for non-pulsed immature DCs (A) and to 200 ng/ml CCL19 or CCL21 for OVA-pulsed DCs (B) was measured. Results are expressed as percentages of basal values obtained without any agonist in DCs of WT mice. Data are mean ± SEM of 5 separate experiments. (C) The expression of CCR7 protein was analysed by flow cytometry. Representative results of four separate experiments in each group are shown. CCR7-protein expression is also expressed as mean fluorescence intensity (MFI) <u>+</u> SEM. (D) The expression of CCR7 mRNA was measured by quantitative real-time TaqMan PCR. Data are mean ± SEM of 9 determinations from three separate experiments. The effects of OGR1 deficiency (WT-OVA vs. OGR1^-/--OVA) were significant throughout the figure (*<i>p</i> < 0.05). </p