Hospitalizations for CM in the US.

<p>A) Hospitalization for CM per million population in HIV-infected and HIV-uninfected patients. B) In hospital mortality per million population in HIV-infected and HIV-uninfected patients.</p

Relative incidence of selected co-morbid conditions among CM hospitalizations compared to all-cause hospitalizations.

<p>Note: SLE =  Systemic Lupus Erythromatosus; RA = Rheumatoid Arthritis; NOS =  Not Otherwise specified.</p><p>Total CM related hospitalizations for the 2008–2009 period: 1,039. Total All hospitalizations for 2008–2009∶33,102,694.</p

Geographic incidence of CM.

<p>A) Map of the US showing, highlighted, states reporting to ARHQ continuously during the study period and their incidence of CM in HIV-infected and B) HIV-uninfected associated hospitalizations per million population.</p

Early Fungicidal Activity as a Candidate Surrogate Endpoint for All-Cause Mortality in Cryptococcal Meningitis: A Systematic Review of the Evidence

<div><p>Background</p><p>Cryptococcal meningitis (CM) is a leading cause of HIV-associated mortality. In clinical trials evaluating treatments for CM, biomarkers of early fungicidal activity (EFA) in cerebrospinal fluid (CSF) have been proposed as candidate surrogate endpoints for all- cause mortality (ACM). However, there has been no systematic evaluation of the group-level or trial-level evidence for EFA as a candidate surrogate endpoint for ACM.</p><p>Methods</p><p>We conducted a systematic review of randomized trials in treatment of CM to evaluate available evidence for EFA measured as culture negativity at 2 weeks/10 weeks and slope of EFA as candidate surrogate endpoints for ACM. We performed sensitivity analysis on superiority trials and high quality trials as determined by Cochrane measures of trial bias.</p><p>Results</p><p>Twenty-seven trials including 2854 patients met inclusion criteria. Mean ACM was 15.8% at 2 weeks and 27.0% at 10 weeks with no overall significant difference between test and control groups. There was a statistically significant group-level correlation between average EFA and ACM at 10 weeks but not at 2 weeks. There was also no statistically significant group-level correlation between CFU culture negativity at 2weeks/10weeks or average EFA slope at 10 weeks. A statistically significant trial-level correlation was identified between EFA slope and ACM at 2 weeks, but is likely misleading, as there was no treatment effect on ACM.</p><p>Conclusions</p><p>Mortality remains high in short time periods in CM clinical trials. Using published data and Institute of Medicine criteria, evidence for use of EFA as a surrogate endpoint for ACM is insufficient and could provide misleading results from clinical trials. ACM should be used as a primary endpoint evaluating treatments for cryptococcal meningitis.</p></div

Trial–level correlations of treatment effect on EFA compared to treatment effects on ACM: All studies with non-missing data are displayed.

<p>N = 9 studies and 26 arms A) Average EFA slope vs ACM at 2 and 10 weeks (6 studies and 18 arms). B) % CSF culture negative vs ACM at 2 and 10 weeks (4 studies and 12 arms).</p

Flow diagram of search strategy and studies selected.

<p>Flow diagram of search strategy and studies selected.</p

Effect of cryptococcal laccase on morphological patterns of pulmonary inflammation and pathological lesions in <i>C. neoformans</i> infected lungs.

<p>Lungs collected from H99-infected (A, C,) and <i>lac1Δ</i>-infected (B, D,) mice were perfused with buffered formalin, fixed, and processed for histology at week 3 postinfection. Representative photomicrographs of H&E + mucicarmine-stained slides taken at 40× (A, B) and 100× (C, D) objective power. Note that sections obtained from wt-infected lungs frequently revealed macrophages harboring intracellular cryptococci encircled by large capsular halos (black arrows) as compared to lung section obtained from <i>Lac1Δ</i>-infected lungs which revealed numerous macrophages with intracellular inclusions consistent with degraded cryptococcal organisms (blue arrows). Also, the extended macrophages in H99-infected mice are heavily laden with YM1/2 crystals (red arrows) which were not observed in <i>lac1Δ</i>-infected lungs. Lastly, note that H99 infection leads to the accumulation of eosinophils (yellow arrow) in the lung, compared to the accumulation of lymphocytes (green arrow) in <i>lac1Δ</i>-infected lungs.</p

Effect of cryptococcal laccase on lung leukocyte cytokine production.

<p>Lung leukocytes were isolated from the lungs of mice infected with <i>C. neoformans</i> strain H99 or <i>lac1</i>Δ at 3 wpi and cultured for 24 h at 5×10⁶ cells/ml. Cytokine levels were evaluated by ELISA in cell culture supernatants. Bars represent mean cytokine concentration ±SEM (pg/ml). Bars represent mean ± SEM from 2 separate matched experiments, <i>N</i> = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant; NS, no significant difference between wt and mutant.</p

Effect of cryptococcal laccase on the recruitment of total lung leukocytes and leukocyte subsets.

<p>Lung leukocytes were isolated from mice infected with H99 or <i>lac1Δ</i>, antibody, stained and analyzed using flow cytometric analysis as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047853#s4" target="_blank">Materials and Methods</a>. The total number of CD45+ leukocytes was determined by multiplying the frequency of CD45+ by total number cells at 2 and 3 wpi (A); Representative FACS plots show the staining strategy to isolate lung leukocyte subsets within gated populations of CD45+ leukocytess. Macrophage, Eosinophil, Neutrophil, and Lymphocyte subsets were identified based on the expression of Gr-1 vs CD11c. Total numbers of Macrophages, Eosinophils, Neutrophils, and Lymphocytse were determined by multiplying the frequency of each subset by the total number of CD45+ leukocytes at 3 wpi (B). Bars represent data (mean ± SEM) from 2–3 separate matched experiments, <i>N</i> = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant; NS, no significant difference between wt and mutant.</p

Effect of cryptococcal laccase on the activation of pulmonary macrophages.

<p>Lung leukocytes were isolated from uninfected mice or mice infected with <i>C. neoformans</i> strain H99 or <i>lac1Δ</i> at 3 wpi. (A) Pulmonary macrophages were purified as described in methods. RNA was extracted, converted to cDNA, and evaluated by qPCR to quantify alternative (Arg1) versus classical (iNOS) macrophage activation gene expression. The data were normalized to GAPDH mRNA levels and expressed as percentage of GAPDH (% GAPDH). (B) Pulmonary macrophages (CD11c+/F4/80+) were gated from CD45+ lung leukocytes by flow cytometry analysis. The activation phenotype of pulmonary macrophages was evaluated by the surface expression of MHC class II (MHC II) and Mac2. Specific antibody staining is depicted as solid lines and isotype-control staining as shaded histograms. The bar graph presents mean fluorescence intensity (MFI) of positive cells derived from these histograms. Note that H99 infection was associated with increased expression of Arg1, compared with macrophages obtained from <i>lac1Δ</i>-infected mice. In contrast, H99 infection was associated with increased expression of Mac2 and decreased expression of MHC II in the pulmonary macrophages, compared with <i>lac1Δ</i> -infected mice. Bars represent data (mean ± SEM) from 2–3 separate matched experiments, <i>N</i> = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant; NS, no significant difference between wt and mutant.</p