Laura K. Najvar

Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus.

ABSTRACT Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The ΔcnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the ΔcnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the ΔcnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the ΔcnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

Disruption of a Nonribosomal Peptide Synthetase in Aspergillus fumigatus Eliminates Gliotoxin Production

ABSTRACT The fungal secondary metabolite gliotoxin produced by Aspergillus fumigatus has been hypothesized to be important in the development of invasive aspergillosis. In this study, we addressed this hypothesis by disrupting a nonribosomal peptide synthetase (NRPS) (encoded by gliP) predicted to be involved in gliotoxin production. Mutants with a disrupted gliP locus failed to produce gliotoxin, which confirmed the role of the NRPS encoded by gliP in gliotoxin biosynthesis. We found no morphological, developmental, or physiological defects in ΔgliP mutant strains. In addition, disruption of gliP resulted in down regulation of gene expression in the gliotoxin biosynthesis gene cluster, which was restored with addition of exogenous gliotoxin. This interesting result suggests a role for gliotoxin in regulating its own production. Culture filtrates from the ΔgliP mutant were unable to inhibit ionomycin-dependent degranulation of mast cells, suggesting a role for gliotoxin in suppressing mast cell degranulation and possibly in disease development. However, the ΔgliP mutant did not have an impact on survival or tissue burden in a murine inhalational model of invasive aspergillosis. This result suggests that gliotoxin is not required for virulence in an immunosuppressed host with an invasive pulmonary infection.

In Vivo Activity of Posaconazole against Mucor spp. in an Immunosuppressed-Mouse Model

ABSTRACT The in vivo activities of posaconazole, itraconazole, and amphotericin B in neutropenic mice with zygomycosis were compared. The in vitro MICs of posaconazole and itraconazole for the strains of Mucor spp. used in this study ranged from 0.125 to 8 μg/ml and 0.25 to 8 μg/ml, respectively. The in vitro MIC range for amphotericin B is 0.125 to 0.25 μg/ml. At twice-daily doses of ≥15 mg/kg of body weight, posaconazole prolonged the survival of the mice and reduced tissue burden.

Caspofungin Resistance in Candida albicans: Correlating Clinical Outcome with Laboratory Susceptibility Testing of Three Isogenic Isolates Serially Obtained from a Patient with Progressive Candida Esophagitis

ABSTRACT A patient with azole-refractory thrush-esophagitis responded initially to caspofungin, but the treatment eventually failed. In a murine model, caspofungin was effective against two early isolates for which the MICs of caspofungin were low, but it was less effective against a late isolate for which the MIC of caspofungin was greater. We concluded that there is a correlation between in vivo failure and rising in vitro caspofungin MICs.

Polymerase Chain Reaction Detection of Aspergillus DNA in Experimental Models of Invasive Aspergillosis

To determine the sensitivity of polymerase chain reaction (PCR) assays for the diagnosis of invasive aspergillosis, results of quantitative culture, PCR-ELISA, and a quantitative LightCycler assay (Roche Diagnostics) of blood and organ specimens of experimentally infected mice and rabbits were compared. By PCR-ELISA, 297 of 379 murine lung specimens were positive, but only 235 of 379 were culture positive. Whereas 64 culture-negative lungs were positive by PCR, Aspergillus was grown from only 2 PCR-negative samples. The PCR assay was 19.4 times more sensitive than culture. None of the 68 blood cultures from mice and rabbits were positive for Aspergillus fumigatus, whereas PCR detected Aspergillus DNA in 17 of 68 blood samples. Quantitative PCR analysis of blood samples showed a fungus load of 10(1)-10(2) cfu/mL of blood. The data confirm the superior sensitivity of PCR for the diagnosis of experimental Aspergillus infections.

Antifungal Activity of Amphotericin B Cochleates against Candida albicans Infection in a Mouse Model

ABSTRACT Cochleates are lipid-based supramolecular assemblies composed of natural products, negatively charged phospholipid, and a divalent cation. Cochleates can encapsulate amphotericin B (AmB), an important antifungal drug. AmB cochleates (CAMB) have a unique shape and the ability to target AmB to fungi. The minimal inhibitory concentration and the minimum lethal concentration against Candida albicans are similar to that for desoxycholate AmB (DAMB; Fungizone). In vitro, CAMB induced no hemolysis of human red blood cells at concentrations of as high as 500 μg of AmB/ml, and DAMB was highly hemolytic at 10 μg of AmB/ml. CAMB protect ICR mice infected with C. albicans when the agent is administered intraperitoneally at doses of as low as 0.1 mg/kg/day. In a tissue burden study, CAMB, DAMB, and AmBisome (liposomal AmB; LAMB) were effective in the kidneys, but in the spleen CAMB was more potent than DAMB at 1 mg/kg/day and was equivalent to LAMB at 10 mg/kg/day. In summary, CAMB are highly effective in treating murine candidiasis and compare well with AmBisome and AmB.

In Vivo Efficacy of Anidulafungin and Caspofungin against Candida glabrata and Association with In Vitro Potency in the Presence of Sera

ABSTRACT In vitro studies have demonstrated that anidulafungin has greater potency than caspofungin against Candida glabrata. However, data from in vivo studies demonstrating that it has superior efficacy are lacking. The objective of this study was to compare the activities of anidulafungin and caspofungin against C. glabrata in a murine model of disseminated candidiasis. Two clinical C. glabrata isolates were used, including one with reduced caspofungin susceptibility. MICs were determined by broth microdilution in the presence and absence of sera. For the animal studies, mice were immunosuppressed with 5-fluorouracil one day prior to intravenous inoculation. Treatment with anidulafungin and caspofungin (0, 0.5, 1, 5, and 10 mg/kg of body weight per day) was begun 24 h later and was continued through day 7 postinoculation. The CFU were enumerated from kidney tissue. According to the standard microdilution methodology, anidulafungin had superior in vitro activity. However, this enhanced potency was attenuated by the addition of mouse and human sera. Caspofungin reduced the kidney fungal burden at lower doses compared to that achieved with anidulafungin in mice infected with the isolate with the lower MIC. Against the strain with the elevated caspofungin MIC, both anidulafungin and caspofungin were effective in reducing the kidney fungal burden at the higher doses studied. Despite the greater in vitro activity of anidulafungin in the absence of sera, both echinocandins were similarly effective in reducing the fungal burden in kidney tissue. The superior in vitro activity of anidulafungin did not confer enhanced in vivo efficacy against C. glabrata.

Correlation between Antifungal Susceptibilities of Coccidioides immitis In Vitro and Antifungal Treatment with Caspofungin in a Mouse Model

ABSTRACT Caspofungin (Merck Pharmaceuticals) was tested in vitro against 25 clinical isolates of Coccidoides immitis. In vitro susceptibility testing was performed in accordance with the National Committee for Clinical Laboratory Standards document M38-P guidelines. Two C. immitis isolates for which the caspofungin MICs were different were selected for determination of the minimum effective concentration (MEC), and these same strains were used for animal studies. Survival and tissue burdens of the spleens, livers, and lungs were used as antifungal response markers. Mice infected with strain 98-449 (48-h MIC, 8 μg/ml; 48-h MEC, 0.125 μg/ml) showed 100% survival to day 50 when treated with caspofungin at ≥1 mg/kg. Mice infected with strain 98-571 (48-h MIC, 64 μg/ml; 48-h MEC, 0.125 μg/ml) displayed ≥80% survival when the treatment was caspofungin at ≥5 mg/kg. Treatment with caspofungin at 0.5, 1, 5, or 10 mg/kg was effective in reducing the tissue fungal burdens of mice infected with either isolate. When tissue fungal burden study results were compared between strains, caspofungin showed no statistically significant difference in efficacy in the organs of the mice treated with both strains. A better in vitro-in vivo correlation was noted when we used the MEC instead of the MIC as the endpoint for antifungal susceptibility testing. Caspofungin may have a role in the treatment of coccidioidomycosis.

In Vitro and In Vivo Activities of Posaconazole against Coccidioides immitis

ABSTRACT Posaconazole (SCH 56592) was tested against 25 strains of Coccidioides immitis to determine their in vitro susceptibilities. The geometric mean 48-h MIC of posaconazole (POSA) was 0.5 μg/ml, the MIC range was 0.25 to 1 μg/ml, and the MIC at which 50% of the isolates tested are inhibited (MIC50) and the MIC90 were 0.5 and 1 μg/ml, respectively. The geometric mean 48-h MIC of itraconazole (ITRA) was 0.23 μg/ml, the MIC range was 0.125 to 0.5 μg/ml, and the MIC50 and MIC90 were both 0.25 μg/ml. Two strains of C. immitis were selected for in vivo studies on the basis of the POSA 48-h MICs for the isolates. POSA orally administered at 0.01, 0.1, 0.5, 1, 5, and 10 mg/kg of body weight/day was compared with ITRA administered at 10 and 30 mg/kg three times a day. The spleens and livers of mice that died or survived to day 50 were removed to measure the fungal burdens. Mice had ≥90% survival when they were treated with ≥0.5 mg of POSA per kg or 30 mg of ITRA per kg. Cultures of whole spleens and livers from mice treated with 10 mg of POSA per kg showed ≥70% sterilization. No sterilization of whole spleens and livers from mice treated with ITRA was seen. POSA displayed potent in vivo activity against the two strains of C. immitis tested.

Assessment of Aspergillus fumigatus Burden in Pulmonary Tissue of Guinea Pigs by Quantitative PCR, Galactomannan Enzyme Immunoassay, and Quantitative Culture

ABSTRACT Early diagnosis of invasive pulmonary aspergillosis is problematic in some patient groups due to the lack of rapid, sensitive, specific, and reliable diagnostic tests. Fungal burden and therapeutic efficacy were assessed by survival, quantitative culture (CFU counts), galactomannan enzyme immunoassay (GM-EIA), and quantitative PCR (qPCR) in a new guinea pig model of invasive pulmonary aspergillosis using an aerosol challenge. At 1 day postinfection, qPCR determined that the pulmonary fungal burden was 2 log10 higher than that determined by CFU counting and increased significantly (P < 0.03) over time. In contrast, the tissue burden assessed by CFU counting did not rise over the course of the study. Therapy with the antifungal drug voriconazole produced statistically significant decreases in pulmonary fungal burden, as detected by CFU counting (P < 0.02), qPCR, and GM-EIA (both P < 0.0002). Daily assessment of the progression of fungal infection in serum was performed by qPCR and GM-EIA. GM-EIA demonstrated a statistically significant reduction in the fungal load on days 6 and 7 in voriconazole-treated animals compared to time-matched controls (P < 0.02). Confirmation of fungal tissue burden by two or more methods should provide a more precise account of the burden, allowing improved assessment of diagnostic and therapeutic strategies in invasive pulmonary aspergillosis.