Lung histology in WT and DPPI^−/− mice on day 3 after oropharyngeal <i>A. fumigatus</i> (1.25×10⁷ conidia per mouse) administration.

<p>In both WT (A) and DPPI^−/− (B) mice, mild predominantly peribronchovascular inflammation occurred (H&E, 100×). No evidence of invasive hyphae was present with GMS staining (400×) in either WT (C) or DPPI^−/− (D) mice. n = 5 mice per genotype.</p

WT mice and NE^−/−×CG^−/− mice were resistant to <i>Burkholderia cepacia</i> infection, whereas p47<i>^phox−/−</i> mice were highly susceptible.

<p>A) Kaplan-Meier survival curves in WT, p47<i>^phox</i>^−/− and NE^−/−×CG^−/− mice administered intraperitoneal <i>B. cepacia</i> (4×10⁷ CFUs/mouse). Log-rank analysis, p<0.0002 comparing WT with p47<i>^phox</i>^−/− mice and p<0.0002 comparing NE^−/−×CG^−/− mice with p47<i>^phox</i>^−/− mice. n = 10 mice per genotype. B) In separate experiments, mice (n = 5 per genotype) were administered the same inoculum of <i>B. cepacia</i>, and quantitative cultures were performed at 24 h. WT and NE^−/−×CG^−/− mice cleared infection, whereas bacterial infection persisted in the peritoneum and spleens of p47<i>^phox</i>^−/− mice. Circles, no growth. *, p<0.03; **, p<0.01.</p

Fungal burden in WT and NE^−/−×CG^−/− mice after <i>A. fumigatus</i> administration.

<p>Mice were administered <i>A. fumigatus</i> (1.25×10⁷ conidia per mouse) by oropharyngeal aspiration and sacrificed on day 3. A) Quantitative fungal cultures in lung homogenates, B) serum galactomannan, C) BALF galactomannan. n = 10 mice per genotype subjected to infection (A. fum), and n = 1 mouse per genotype subjected to sham-infection. No significant differences occurred in quantitative lung fungal cultures, serum galactomannan, and BALF galactomannan between <i>Aspergillus</i>-infected WT mice and NE^−/−×CG^−/− mice.</p

Lung histology and airway inflammation in WT and NE^−/−×CG^−/− mice after <i>A. fumigatus</i> administration.

<p>Mice were administered <i>A. fumigatus</i> (1.25×10⁷ conidia per mouse) by oropharyngeal aspiration and sacrificed on day 3. A) BALF leukocyte recovery and B) percent lung inflammation were similar in WT and NE^−/−×CG^−/− mice. Representative lung histology from WT (C and D) and NE^−/−×CG^−/− mice (E and F). Predominantly peribronchovascular neutrophilic and lymphohistiocytic inflammation occurred in both genotypes (C and E; H&E, 40×). GMS staining (400×) of lung sections from WT (D) and NE^−/−×CG^−/− (F) mice showed what appeared to be degenerated hyphal fragments, but no evidence of intact invasive hyphae. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028149#s2" target="_blank">Results</a> are representative of 15 WT and 10 NE^−/−×CG^−/− mice. By comparison, p47<i>^phox−/−</i> mice administered <i>A. fumigatus</i> at 0.1% of this inoculum (1.25×10⁴ conidia per mouse) and sacrificed on day 3 had evidence of fungal pneumonia characterized by G) multiple foci of neutrophilic consolidation (H&E, 40×), and H) hyphal parenchymal invasion (arrow) (GMS, 400×).</p

(a) Mean Z-scores for the time-weighted averages of galactomannan and beta-D-glucan composite values, and (b) time-weighted averages for beta-D-glucan values, galactomannan values, and galactomannan optical density index, in responders vs non-responders at week 6 and week 12.

<p>GM: Galactomannan, BDG: Beta-D-Glucan, GMI: Galactomannan Optical Density Index, W: Week, R: Responder, NR: Non-responder, N: Number, CI: Confidence Interval.</p><p>^<b>a</b> Some patients did not have data available for both biomarkers throughout the study; therefore, the numbers of patients with BDG, GM and/or GMI at weeks 2 and 6 differ from the total number of patients in the BDG and GM column.</p><p>(a) Mean Z-scores for the time-weighted averages of galactomannan and beta-D-glucan composite values, and (b) time-weighted averages for beta-D-glucan values, galactomannan values, and galactomannan optical density index, in responders vs non-responders at week 6 and week 12.</p

Association of galactomannan assay optical density index (GMI) as a dichotomous variable (positive [GMI ≥ 0.5] vs negative) between baseline and week 2 of treatment with clinical response and survival at 6 and 12 weeks.

<p>GMI: Galactomannan Optical Density Index, OR: Odds Ratio, CI: Confidence Interval</p><p>^a Clinical response: complete or partial response, as described in Methods [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129022#pone.0129022.ref005" target="_blank">5</a>].</p><p>^b Only one patient had positive GMI at baseline and negative GMI by week 2, so this category (+/-) was not included in the analysis.</p><p>^c Due to low number of observations, there was lack of model convergence and no results were generated.</p><p>Association of galactomannan assay optical density index (GMI) as a dichotomous variable (positive [GMI ≥ 0.5] vs negative) between baseline and week 2 of treatment with clinical response and survival at 6 and 12 weeks.</p

Correlation between Circulating Fungal Biomarkers and Clinical Outcome in Invasive Aspergillosis

<div><p>Objective means are needed to predict and assess clinical response in patients treated for invasive aspergillosis (IA). We examined whether early changes in serum galactomannan (GM) and/or β-D-glucan (BDG) can predict clinical outcomes. Patients with proven or probable IA were prospectively enrolled, and serial GM and BDG levels and GM optical density indices (GMI) were calculated twice weekly for 6 weeks following initiation of standard-of-care antifungal therapy. Changes in these biomarkers during the first 2 and 6 weeks of treatment were analyzed for associations with clinical response and survival at weeks 6 and 12. Among 47 patients with IA, 53.2% (25/47) and 65.9% (27/41) had clinical response by weeks 6 and 12, respectively. Changes in biomarkers during the first 2 weeks were associated with clinical response at 6 weeks (GMI, <i>P</i> = 0.03) and 12 weeks (GM+BDG composite, <i>P</i> = 0.05; GM, <i>P</i> = 0.04; GMI, <i>P</i> = 0.02). Changes in biomarkers during the first 6 weeks were also associated with clinical response at 6 weeks (GM, <i>P</i> = 0.05; GMI, <i>P</i> = 0.03) and 12 weeks (BDG+GM, <i>P</i> = 0.02; GM, <i>P</i> = 0.02; GMI, <i>P</i> = 0.01). Overall survival rates at 6 weeks and 12 weeks were 87.2% (41/47) and 79.1% (34/43), respectively. Decreasing biomarkers in the first 2 weeks were associated with survival at 6 weeks (BDG+GM, <i>P</i> = 0.03; BDG, <i>P</i> = 0.01; GM, <i>P</i> = 0.03) and at 12 weeks (BDG+GM, <i>P</i> = 0.01; BDG, <i>P</i> = 0.03; GM, <i>P</i> = 0.01; GMI, <i>P</i> = 0.007). Similar correlations occurred for biomarkers measured over 6 weeks. Patients with negative baseline GMI and/or persistently negative GMI during the first 2 weeks were more likely to have CR and survival. These results suggest that changes of biomarkers may be informative to predict and/or assess response to therapy and survival in patients treated for IA.</p></div

Patient baseline characteristics.

<p>IA: Invasive Aspergillosis, GMI: Galactomannan Optical Density Index, BDG: β-D-glucan.</p><p>^aUnderlying disease, comorbidities, and microbiologic diagnostic tests were not mutually exclusive.</p><p>^bAdministration of corticosteroids (0.3 mg/kg/day of prednisone equivalent) for a minimum of 3 weeks prior to enrollment.</p><p>^cFour patients with proven IA had a positive culture for <i>Aspergillus</i> species: 3 from normally sterile sites and 1 from a sputum/bronchoalveolar lavage culture.</p><p>^dPatients with positive GM and BDG as tested at the institutions they were enrolled at are included in this Table. Some of them had negative GM and BDG when tested at the central laboratory for the purposes of this study.</p><p>^eOne patient had both sinusitis and lower tract respiratory involvement.</p><p>^fSix and 2 patients were treated with liposomal amphotericin B and amphotericin B deoxycholate, respectively.</p><p>^gOne patient was treated with fluconazole alone and the other patient received blinded treatment with a mould acting agent as part of a clinical trial.</p><p>^hPatients received more than one agent concomitantly: echinocandin and voriconazole (n = 2), echinocandin and amphotericin B deoxycholate (n = 1), and voriconazole and amphotericin B deoxycholate (n = 1).</p><p>Patient baseline characteristics.</p