Defining Responses to Therapy and Study Outcomes in Clinical Trials of Invasive Fungal Diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer Consensus Criteria

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledg

Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox−/−) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)−/−×cathepsin G (CG)−/− mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox−/− mice, whereas NE−/−×CG−/− mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens

In Vitro Susceptibilities of 217 Clinical Isolates of Zygomycetes to Conventional and New Antifungal Agents▿

We evaluated the in vitro susceptibilities of 217 zygomycetes to amphotericin B, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, caspofungin, and flucytosine. The significant in vitro activity of posaconazole against several species appears to support its reported clinical efficacy. Decreased susceptibility to amphotericin B was noted with Cunninghamella bertholletiae

Serum IL-12:IL-6 Ratio Reliably Distinguishes Infectious from Non-Infectious Causes of Fever during Autologous Stem Cell Transplantation (ASCT)

Abstract Fever occurs commonly in patients (pts) undergoing ASCT, both during neutropenia and after neutrophil engraftment, but infectious etiologies are infrequently identified. A reliable and rapid method to distinguish infectious from non-infectious etiologies of fever may be able to reduce unnecessary diagnostic testing and empiric antimicrobial administration, leading to decreased costs, duration of hospital stays, and treatment related complications. We hypothesized that the serum cytokine profile during fever due to clinically significant infection is distinct from that during non-infectious febrile episodes. In a prospective exploratory study we assessed serum IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, TNFα, and IFNγ levels by sandwich ELISA in 65 pts undergoing ASCT for non-Hodgkin’s lymphoma (n=20), Hodgkin’s disease (n=22), multiple myeloma (n=14), or AL amyloidosis (n=9). Cytokine levels were measured at multiple pre-defined times during ASCT; the results presented here are based on measurements upon admission, during the first episode of febrile neutropenia, during the first episode of fever within 72 hours after neutrophil engraftment, and upon discharge. Standard evaluation of fever included chest x-rays and blood and urine cultures. Other diagnostic testing, eg stool cultures and CT scans, were performed at the discretion of the responsible physicians as deemed appropriate by patients’ symptoms. 69 febrile episodes occurred in 54 pts: 39 episodes occurred during neutropenia (12 due to documented infections); 30 episodes occurred after engraftment, 17 of which were preceded within 72 hours by fever during neutropenia (5 infectious, 12 non-infectious). Only 2 of the 13 new-onset post-engraftment fevers had an identifiable infectious etiology. Levels of IL-1β, IL-2, IL-8, IL-10, TNFα, and IFNγ did not distinguish infectious from non-infectious etiologies of fever. Mean IL-12 levels at the time of fever were significantly higher among pts without infection compared to pts with infection, regardless of neutrophil count; conversely, mean IL-6 levels were lower in febrile pts without infection. Compared to febrile pts with infection, the mean IL-12:IL-6 ratio was significantly higher in febrile pts without infection, both during neutropenia and after neutrophil engraftment. IL-12 and IL-6 levels and IL-12:IL-6 ratio did not vary significantly by histology, either at diagnosis or at discharge. IL-12:IL-6 ratio during fever Febrile period Infection No infection P-value Neutropenia 1.0 58.7 <0.001 Post-engraftment 5.28 84.6 0.01 The IL-12:IL-6 ratio on the day of neutrophil engraftment in 11 pts without fever during the entire ASCT course was similar to that of normal healthy volunteers (904.6 vs. 708.0, P=NS) and significantly higher than among pts with infection during neutropenia (P<0.001) or after engraftment (P=0.006). These data suggest that the IL-12:IL-6 ratio reliably distinguishes infectious from non-infectious etiologies of fever during the course of ASCT. Most febrile episodes that occur after neutrophil engraftment are not due to infection. Prospective studies to determine the sensitivity and specificity of an IL-12:IL-6 ratio threshold that distinguishes infectious from non-infectious causes of fever in the transplant and non-transplant settings are warranted

Correlation between Circulating Fungal Biomarkers and Clinical Outcome in Invasive Aspergillosis

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, P = 0.03) and 12 weeks (GM+BDG composite, P = 0.05; GM, P = 0.04; GMI, P = 0.02). Changes in biomarkers during the first 6 weeks were also associated with clinical response at 6 weeks (GM, P = 0.05; GMI, P = 0.03) and 12 weeks (BDG+GM, P = 0.02; GM, P = 0.02; GMI, P = 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, P = 0.03; BDG, P = 0.01; GM, P = 0.03) and at 12 weeks (BDG+GM, P = 0.01; BDG, P = 0.03; GM, P = 0.01; GMI, P = 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

Correlation between Circulating Fungal Biomarkers and Clinical Outcome in Invasive Aspergillosis

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, P = 0.03) and 12 weeks (GM+BDG composite, P = 0.05; GM, P = 0.04; GMI, P = 0.02). Changes in biomarkers during the first 6 weeks were also associated with clinical response at 6 weeks (GM, P = 0.05; GMI, P = 0.03) and 12 weeks (BDG+GM, P = 0.02; GM, P = 0.02; GMI, P = 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, P = 0.03; BDG, P = 0.01; GM, P = 0.03) and at 12 weeks (BDG+GM, P = 0.01; BDG, P = 0.03; GM, P = 0.01; GMI, P = 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.status: publishe

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

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