Michail S. Lionakis

Zygomycosis in a Tertiary-Care Cancer Center in the Era of Aspergillus-Active Antifungal Therapy: A Case-Control Observational Study of 27 Recent Cases

BACKGROUND Anecdotal evidence suggests a rise in zygomycosis in association with voriconazole (VRC) use in immunosuppressed patients. METHODS We performed prospective surveillance of patients with zygomycosis (group A; n = 27) and compared them with contemporaneous patients with invasive aspergillosis (group B; n = 54) and with matched contemporaneous high-risk patients without fungal infection (group C; n = 54). We also performed molecular typing and in vitro susceptibility testing of Zygomycetes isolates. RESULTS Nearly all patients with zygomycosis either had leukemia (n = 14) or were allogeneic bone marrow transplant recipients (n = 13). The Zygomycetes isolates (74% of which were of the genus Rhizopus) had different molecular fingerprinting profiles, and all were VRC resistant. In multivariate analysis of groups A and C, VRC prophylaxis (odds ratio [OR], 10.37 [95% confidence interval [CI]], 2.76-38.97]; P = .001), diabetes (OR, 8.39 [95% CI, 2.04-34.35]; P = .003), and malnutrition (OR, 3.70 [95% CI, 1.03-13.27]; P = .045) were found to be independent risk factors for zygomycosis. Between patients with zygomycosis (after excluding 6 patients with mixed mold infections) and patients with aspergillosis, VRC prophylaxis (OR, 20.30 [95% CI, 3.85-108.15]; P = .0001) and sinusitis (OR, 76.72 [95% CI, 6.48-908.15]; P = .001) were the only factors that favored the diagnosis of zygomycosis. CONCLUSIONS Zygomycosis should be considered in immunosuppressed patients who develop sinusitis while receiving VRC prophylaxis, especially those with diabetes and malnutrition.

Glucocorticoids and invasive fungal infections

Since the 1990s, opportunistic fungal infections have emerged as a substantial cause of morbidity and mortality in profoundly immunocompromised patients. Hypercortisolaemic patients, both those with endogenous Cushings syndrome and, much more frequently, those receiving exogenous glucocorticoid therapy, are especially at risk of such infections. This vulnerability is attributed to the complex dysregulation of immunity caused by glucocorticoids. We critically review the spectrum and presentation of invasive fungal infections that arise in the setting of hypercortisolism, and the ways in which glucocorticoids contribute to their pathogenesis. A better knowledge of the interplay between glucocorticoid-induced immunosuppression and invasive fungal infections should assist in earlier recognition and treatment of such infections. Efforts to decrease the intensity of glucocorticoid therapy should help to improve outcomes of opportunistic fungal infections.

Infections Due to Aspergillus terreus: A Multicenter Retrospective Analysis of 83 Cases

Current in vitro and in vivo data indicate that invasive aspergillosis due to Aspergillus terreus is resistant to treatment with amphotericin B. Because little clinical data are available to guide therapy, we performed a retrospective cohort study of cases of invasive A. terreus infections from 1997-2002 to determine whether the use of voriconazole, compared with use of other antifungal therapies, led to an improved patient outcome. We analyzed a total of 83 cases of proven or probable invasive A. terreus infection (47% and 53%, respectively). A total of 66.3% of patients (55 of 83) died during management of IA, with 55.8% mortality (19 of 34 patients) in the voriconazole group and 73.4% mortality (36 of 49) in the group that received therapy with other antifungals. By use of Cox proportional hazards modeling, decreased mortality at 12 weeks was observed in those patients who received voriconazole (hazard ratio, 0.29; 95% CI, 0.15-0.56). Voriconazole is likely to be a better treatment choice for A. terreus infection than is a polyene.

Predictors of Pulmonary Zygomycosis versus Invasive Pulmonary Aspergillosis in Patients with Cancer

BACKGROUND Pulmonary zygomycosis (PZ), an emerging mycosis among patients with cancer, has a clinical manifestation similar to that of invasive pulmonary aspergillosis (IPA). Most cases of PZ in such patients develop as breakthrough infections if treatment with antifungal agents effective against Aspergillus species is administered. However, clinical criteria to differentiate PZ from IPA are lacking. METHODS We retrospectively reviewed the clinical characteristics and computed tomography (CT) findings for 16 patients with cancer and PZ and for 29 contemporaneous patients with cancer and IPA at the time of infection onset (2002-2004). Patients with mixed infections were excluded. Parameters predictive of PZ by univariate analysis were included in a logistic regression model. RESULTS Almost all patients with PZ (15 of 16) and IPA (28 of 29) had underlying hematological malignancies and typical risk factors for invasive mold infections. In logistic regression analysis of clinical characteristics, concomitant sinusitis (odds ratio [OR], 25.7; 95% confidence interval [CI], 1.47-448.15; P = .026) and voriconazole prophylaxis (OR, 7.76; 95% CI, 1.32-45.53; P = .023) were significantly associated with PZ. The presence of multiple (> or = 10) nodules (OR, 19.8; 95% CI, 1.94-202.29; P = .012) and pleural effusion (OR, 5.07; 95% CI, 1.06-24.23; P = .042) at the time that the patient underwent the initial CT were both independent predictors of PZ in the logistic regression analysis of radiological parameters. No difference occurred in the frequency of other CT findings suggestive of pulmonary mold infections (e.g., masses, cavities, halo sign, or air-crescent sign) between the 2 patient groups. CONCLUSIONS PZ in immunocompromised patients with cancer could potentially be distinguished from IPA on the basis of clinical and radiological parameters; prospective validation is needed.

Organ-Specific Innate Immune Responses in a Mouse Model of Invasive Candidiasis

In a fatal mouse model of invasive candidiasis (IC), fungal burden changes with variable dynamics in the kidney, brain, spleen, and liver and declines in all organs except for the kidney, which inexorably loses function. Since leukocytes are required to control Candida, we hypothesized that differential leukocyte infiltration determines organ-specific outcome of the infection. We defined leukocyte accumulation in the blood, kidney, brain, spleen, and liver after infection using fluorescent-activated cell sorting (FACS) and immunohistochemistry. Accumulation of Ly6cintCD11b+ neutrophils predominated in all organs except the brain, where CD45intCD11b+CD11c– microglia were the major leukocytes detected, surrounding foci of invading Candida. Significantly more neutrophils accumulated in the spleen and liver than in the kidney during the first 24 h after infection, when neutrophil presence is critical for Candida control. Conversely, at later time points only the kidney continued to accumulate neutrophils, associated with immunopathology and organ failure. The distribution of neutrophils was completely different in each organ, with large abscesses exclusively forming in the kidney. Candida filamentation, an essential virulence factor, was seen in the kidney but not in the spleen or liver. IC induced Ly6chiCD11b+ inflammatory monocyte and NK1.1+ cell expansion in the blood and all organs tested, and MHCII+F4/80+CD11c– macrophage accumulation, mainly in the spleen and liver. This study is the first detailed analysis of leukocyte subsets accumulating in different target organs during IC. The results delineate immune responses to the same pathogen that are highly idiosyncratic for each organ tested. The work provides novel insights into the balance between effective host defense and immunopathology in IC.

CX3CR1-dependent renal macrophage survival promotes Candida control and host survival

Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX3CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1-/- mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1-/- mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX3CR1 allele CX3CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX3CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation‐based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT . In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence‐activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments.

Frequency and Species Distribution of Gliotoxin-Producing Aspergillus Isolates Recovered from Patients at a Tertiary-Care Cancer Center

ABSTRACT Aspergillus isolates (n = 103) collected from cancer patients were screened to determine the taxonomic distribution and quantity of gliotoxin production. Gliotoxin was detected in 93% of Aspergillus fumigatus, 75% of A. niger, 25% of A. terreus, and 4% of A. flavus cultures. Gliotoxin concentrations were highest in cultures of A. fumigatus.

Drosophila melanogaster as a Facile Model for Large-Scale Studies of Virulence Mechanisms and Antifungal Drug Efficacy in Candida Species

Candida species are the predominant fungal pathogens in humans and an important cause of mortality in immunocompromised patients. We developed a model of candidiasis in Toll (Tl)-deficient Drosophila melanogaster. Similar to the situation in humans, C. parapsilosis was less virulent than C. albicans when injected into Tl mutant flies. In agreement with findings in the mouse model of invasive candidiasis, cph1/cph1 and efg1/efg1 C. albicans mutants had attenuated virulence, and the efg1/efg1 cph1/cph1 double mutant was almost avirulent in Tl mutant flies. Furthermore, the conditional tet-NRG1 C. albicans strain displayed significantly attenuated virulence in flies fed food without doxycycline; virulence was restored to wild-type levels when the strain was injected into Tl mutant flies fed doxycycline-containing food. Fluconazole (FLC) mixed into food significantly protected Tl mutant flies injected with FLC-susceptible C. albicans strains, but FLC had no activity in flies injected with FLC-resistant C. krusei strains. The D. melanogaster model is a promising minihost model for large-scale studies of virulence mechanisms and antifungal drug activity in candidiasis.

Candida and host determinants of susceptibility to invasive candidiasis

Candida is the most common human fungal pathogen and the cause of invasive candidiasis, the fourth leading cause of nosocomial bloodstream infection in the United States with an estimated annual cost of ∼US