Search

Mice were immunosuppressed using corticosteroids and infected with conidia of Aspergillus fumigatus. Beginning 1 day after injection, mice were treated orally either with Noble agar or with SCH56592 suspended in Noble agar, or intraperitoneally with amphotericin B. SCH56592 prolonged survival and reduced lung tissue counts of A. fumigatus, while amphotericin B had marginal benefit. SCH56592 merits further development for treatment of aspergillosis. [ABSTRACT FROM PUBLISHER]

We evaluated the in vivo activity of nikkomycin Z against central nervous system coccidioidomycosis. Mice were inoculated intracranially with arthroconidia of Coccidoides immitis , and treatment with nikkomycin Z (50, 100, or 300 mg/kg orally TID) or fluconazole (25 mg/kg orally BID) began 2 days later. Each dose of nikkomycin Z and fluconazole significantly improved survival and reduced brain fungal burden compared with vehicle control. Further studies of nikkomycin Z against coccidioidomycosis are warranted., Importance: Coccidioides species are endemic fungi that are capable of causing disease in patients with various comorbidities, as well as in otherwise healthy individuals. Treatment options for coccidioidomycosis are suboptimal, as azole antifungals may be limited by drug interactions and adverse effects due to interactions with enzymes found in humans and other mammals. Nikkomycin Z is an investigational agent that works against a target specific to the fungal cell wall (chitin), which is not present in the cells of humans or other mammals. In this study, we show that frequent oral administration of nikkomycin Z is effective in an experimental model of central nervous system coccidioidomycosis. Further studies of nikkomycin Z against coccidioidomycosis may be warranted., Competing Interests: N.P.W. has received research support from the UT Health San Antonio from bioMerieux, F2G, Mycovia, Sfunga, and Scynexis and has served on an advisory board for F2G. D.J.L. is employed by Valley Fever Solutions. T.F.P. has served as a consultant and/or received research support from Cidara, F2G, Gilead, Scynexis, and Elion Therapeutics.

Ibrexafungerp (formerly SCY-078) is the first member of the triterpenoid class that prevents the synthesis of the fungal cell wall polymer β-(1,3)-D-glucan by inhibiting the enzyme glucan synthase. We evaluated the in vivo efficacy of ibrexafungerp against pulmonary mucormycosis using an established murine model. Neutropenic mice were intratracheally infected with either Rhizopus delemar or Mucor circinelloides . Treatment with placebo (diluent control), ibrexafungerp (30 mg/kg, PO BID), liposomal amphotericin B (LAMB 10 mg/kg IV QD), posaconazole (PSC 30 mg/kg PO QD), or a combination of ibrexafungerp plus LAMB or ibrexafungerp plus PSC began 16 h post-infection and continued for 7 days for ibrexafungerp or PSC and through day 4 for LAMB. Ibrexafungerp was as effective as LAMB or PSC in prolonging median survival (range: 15 days to >21 days) and enhancing overall survival (30%-65%) vs placebo (9 days and 0%; P < 0.001) in mice infected with R. delemar . Furthermore, median survival and overall percent survival resulting from the combination of ibrexafungerp plus LAMB were significantly greater compared to all monotherapies ( P ≤ 0.03). Similar survival results were observed in mice infected with M. circinelloides . Monotherapies also reduce the lung and brain fungal burden by ~0.5-1.0log 10 conidial equivalents (CE)/g of tissue vs placebo in mice infected with R. delemar ( P < 0.05), while a combination of ibrexafungerp plus LAMB lowered the fungal burden by ~0.5-1.5log 10 CE/g compared to placebo or any of the monotherapy groups ( P < 0.03). These results are promising and warrant continued investigation of ibrexafungerp as a novel treatment option against mucormycosis., Competing Interests: A.S.I. has received research support from and served on advisory boards for Astellas, Basilea, Cidara, Matinas, Navigen, Pfizer, and Sfunga. N.P.W. has received support (financial and material) from bioMerieux, Bruker, F2G, Maxwell Biosciences, Mycovia, and Sfunga and has served on an advisory board for F2G. K.B.-E. is an employee of Scynexis. All other authors declare no conflict of interest.

Candidiasis is one of the most frequent nosocomial infections affecting an increasing number of at-risk patients. Candida albicans remains the most frequent causative agent of candidiasis, but, in the last decade, C. auris has emerged as a formidable multi-drug-resistant pathogen. Both species are fully capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. Here, we have screened the Repurposing Hub library from the Broad Institute, containing over 6000 compounds, in search for inhibitors of C. albicans and C. auris biofilm formation. The primary screen identified 57 initial hits against C. albicans and 33 against C. auris . Confirmatory concentration-dependent assays were used to validate the activity of the initial hits and, at the same time, establish their anti-biofilm potency. Based on these results, ebselen, temsirolimus, and compound BAY 11-7082 emerged as the leading repositionable compounds. Subsequent experiments established their spectrum of antifungal activity against yeasts and filamentous fungi. In addition, their in vivo activity was examined in the murine models of hematogenously disseminated C. albicans and C. auris infections. Although promising, further in vitro and in vivo studies are needed to confirm their potential use for the therapy of candidiasis and possibly other fungal infections.

Candida auris is an emerging pathogen that has rapidly spread to many countries on multiple continents. Invasive infections caused by this species are associated with significant mortality, and treatment options are limited due to antifungal resistance. Ibrexafungerp is the first-in-class member of the triterpenoids, which inhibit the production of (1,3)-β-d-glucan and can be administered orally. We evaluated the in vitro activity and in vivo efficacy of ibrexafungerp against C. auris Antifungal susceptibility was tested by broth microdilution against 54 C. auris isolates. Neutropenic mice were intravenously infected with a clinical isolate, and a 7-day treatment course was begun 24 h postinoculation with vehicle control, ibrexafungerp (20, 30, and 40 mg/kg orally twice daily), fluconazole (20 mg/kg orally once daily), or caspofungin (10 mg/kg intraperitoneally once daily). Fungal burden was assessed by colony counts in the kidneys on day 8 and on day 21 or as mice became moribund in the survival arm. Ibrexafungerp demonstrated consistent activity, with MICs ranging between 0.25 and 2 μg/ml against all isolates. Marked improvements in survival were observed in mice treated with the higher doses of ibrexafungerp and caspofungin. Similarly, reductions in kidney fungal burden were also observed in these groups. No improvements in survival or reductions in fungal burden were observed with fluconazole, consistent with the in vitro resistance of the isolate used to establish infection to this azole. These results demonstrate that ibrexafungerp is effective in vivo against C. auris even when the start of therapy is delayed., (Copyright © 2021 American Society for Microbiology.)

There is increased concern that the quality, generalizability and reproducibility of biomedical research can be influenced by the sex of animals used. We studied the differences between male and female mice in response to invasive pulmonary mucormycosis including susceptibility to infection, host immune reaction and responses to antifungal therapy. We used diabetic ketoacidotic (DKA) or neutropenic mice infected with either Rhizopus delemar or Mucor circinelloides . The only difference detected was that when DKA mice were infected with M. circinelloides , female mice were more resistant to infection than male mice (median survival time of 5 vs. 2 days for female and male mice, respectively). However, a 100% lethality was detected among infected animals of both sexes. Treatment with either liposomal amphotericin B (L-AMB) or posaconazole (POSA) protected mice from infection and eliminated the difference seen between infected but untreated female and male mice. Treatment with L-AMB consistently outperformed POSA in prolonging survival and reducing tissue fungal burden of DKA and neutropenic mice infected with R. delemar or M. circinelloides , in both mouse sexes. While little difference was detected in cytokine levels among both sexes, mucormycosis infection in the DKA mouse model induced more inflammatory cytokines/chemokines involved in neutrophil (CXCL1) and macrophage (CXCL2) recruitment vs. uninfected mice. As expected, this inflammatory response was reduced in the neutropenic mouse model. Our studies show that there are few differences between female and male DKA or neutropenic mice infected with mucormycosis with no effect on the outcome of treatment or host immune response.

The in vitro and in vivo activity of the arylamidine T-2307 against Candida auris was evaluated. T-2307 demonstrated in vitro activity (MIC ranges ≤ 0.008 to 0.015 μg/ml at 50% inhibition; 0.125 to >4 μg/ml at 100% inhibition). Treatment with T-2307 (3 mg/kg subcutaneous [SC] once daily) also significantly improved survival (70% at 21 days postinfection) and reduced kidney fungal burden (5.06 log 10 CFU/g) compared to control (0% survival and 7.09 log 10 CFU/g) ( P < 0.01)., (Copyright © 2020 American Society for Microbiology.)

The emerging pathogenic yeast Candida auris is associated with antifungal resistance and high mortality. The novel antifungal agent manogepix (APX001A) inhibits glycosylphosphatidylinositol-anchored protein maturation and has demonstrated activity against numerous pathogenic fungi, including C. auris Our objective was to evaluate the in vivo efficacy of fosmanogepix, the N -phosphonooxymethyl prodrug (APX001), following delayed initiation of therapy in a murine model of C. auris invasive candidiasis. Neutropenic mice were intravenously infected with a fluconazole-resistant clinical isolate of C. auris Twenty-four hours postinoculation, treatment began with vehicle control, fosmanogepix (104 and 130 mg/kg of body weight by intraperitoneal injection three times daily, or intraperitoneal 260 mg/kg twice daily), fluconazole (20 mg/kg by oral gavage once daily), or caspofungin (intraperitoneal 10 mg/kg once daily) and continued for 7 days. Fungal burden was assessed via colony count in the kidneys and brains on day 8 in the fungal burden arm and on day 21 as the mice became moribund in the survival arm. Significant improvements in survival were observed in each group administered fosmanogepix and caspofungin. Similarly, reductions in fungal burden were also observed in both the kidneys and brains of mice treated with the highest dose of fosmanogepix in the fungal burden arm and in each fosmanogepix group and with caspofungin in the survival arm. In contrast, no improvements in survival or reductions in fungal burden were observed in mice treated with fluconazole. These results demonstrate that fosmanogepix is effective in vivo against fluconazole-resistant C. auris even when therapy is delayed., (Copyright © 2019 Wiederhold et al.)

We evaluated extended-interval dosing of the investigational echinocandin rezafungin (1, 4, and 16 mg/kg on days 1, 4, and 7 postinoculation) for the treatment of disseminated invasive aspergillosis caused by azole-resistant Aspergillus fumigatus Survival was significantly improved in mice treated with each dose of rezafungin and supratherapeutic posaconazole (20 mg/kg twice daily). Kidney fungal burden, as measured by quantitative real-time PCR, was also significantly reduced in mice treated with rezafungin although variability was observed., (Copyright © 2019 Wiederhold et al.)

Candida auris is an emerging pathogen associated with significant mortality and often multidrug resistance. VT-1598, a tetrazole-based fungal CYP51-specific inhibitor, was evaluated in vitro and in vivo against C. auris Susceptibility testing was performed against 100 clinical isolates of C. auris by broth microdilution. Neutropenic mice were infected intravenously with C. auris , and treatment began 24 h postinoculation with a vehicle control, oral VT-1598 (5, 15, and 50 mg/kg of body weight once daily), oral fluconazole (20 mg/kg once daily), or intraperitoneal caspofungin (10 mg/kg once daily), which continued for 7 days. Fungal burden was assessed in the kidneys and brains on day 8 in the fungal burden arm and on the days the mice succumbed to infection or on day 21 in the survival arm. VT-1598 plasma trough concentrations were also assessed on day 8. VT-1598 demonstrated in vitro activity against C. auris , with a mode MIC of 0.25 μg/ml and MICs ranging from 0.03 to 8 μg/ml. Treatment with VT-1598 resulted in significant and dose-dependent improvements in survival (median survival, 15 and >21 days for VT-1598 at 15 and 50 mg/kg, respectively) and reductions in kidney and brain fungal burden (reductions of 1.88 to 3.61 log 10 CFU/g) compared to the control (5 days). The reductions in fungal burden correlated with plasma trough concentrations. Treatment with caspofungin, but not fluconazole, also resulted in significant improvements in survival and reductions in fungal burden compared to those with the control. These results suggest that VT-1598 may be a future option for the treatment of invasive infections caused by C. auris ., (Copyright © 2019 American Society for Microbiology.)

VT-1129 is a novel fungal enzyme-specific Cyp51 inhibitor with potent cryptococcal activity. Because of its long half-life (>6 days in mice) and our desire to quickly reach potent efficacy, we evaluated a VT-1129 loading dose-maintenance dose strategy against cryptococcal meningitis. VT-1129 plasma and brain pharmacokinetics were first studied in healthy mice, and these data were used to model loading dose-maintenance dose regimens to generate different steady-state concentrations. Mice were inoculated intracranially with Cryptococcus neoformans , and oral treatment began 1 day later. Treatment consisted of placebo or one of three VT-1129 loading dose-maintenance dose regimens, i.e., loading dose of 1, 3, or 30 mg/kg on day 1, followed by once-daily maintenance doses of 0.15, 0.5, or 5 mg/kg, respectively. In the fungal burden arm, therapy continued for 14 days and brains were collected on day 15 for fungal burden assessments. In the survival arm, treatment continued for 10 days, after which mice were monitored without therapy until day 30. VT-1129 plasma and brain concentrations were also measured. All VT-1129 doses significantly improved survival and reduced fungal burdens, compared to placebo. VT-1129 plasma and brain levels correlated with fungal burden reductions ( R 2 = 0.72 and R 2 = 0.67, respectively), with a plasma concentration of 1 μg/ml yielding a reduction of ∼5 log 10 CFU/g. With the highest loading dose-maintenance dose regimen, fungal burdens were undetectable in one-half of the mice in the fungal burden arm and in one-fourth of the mice in the survival arm, 20 days after the final dose. These data support a loading dose-maintenance dose strategy for quickly reaching highly efficacious VT-1129 concentrations for treating cryptococcal meningitis., (Copyright © 2018 American Society for Microbiology.)

Cryptococcal meningitis is a significant cause of morbidity and mortality in immunocompromised patients. VT-1129 is a novel fungus-specific Cyp51 inhibitor with potent in vitro activity against Cryptococcus species. Our objective was to evaluate the in vivo efficacy of VT-1129 against cryptococcal meningitis. Mice were inoculated intracranially with Cryptococcus neoformans Oral treatment with VT-1129, fluconazole, or placebo began 1 day later and continued for either 7 or 14 days, and brains and plasma were collected on day 8 or 15, 1 day after therapy ended, and the fungal burden was assessed. In the survival study, treatment continued until day 10 or day 28, after which mice were monitored off therapy until day 30 or day 60, respectively, to assess survival. The fungal burden was also assessed in the survival arm. VT-1129 plasma and brain concentrations were also measured. VT-1129 reached a significant maximal survival benefit (100%) at a dose of 20 mg/kg of body weight once daily. VT-1129 at doses of ≥0.3 mg/kg/day and each dose of fluconazole significantly reduced the brain tissue fungal burden compared to that in the control after both 7 and 14 days of dosing. The fungal burden was also undetectable in most mice treated with a dose of ≥3 mg/kg/day, even ≥20 days after dosing had stopped, in the survival arm. In contrast, rebounds in fungal burden were observed with fluconazole. These results are consistent with the VT-1129 concentrations, which remained elevated long after dosing had stopped. These data demonstrate the potential utility of VT-1129 to have a marked impact in the treatment of cryptococcal meningitis., (Copyright © 2018 American Society for Microbiology.)

Olorofim (formerly F901318) is an advanced analog of the orotomide class that inhibits fungal pyrimidine biosynthesis. We evaluated the in vitro and in vivo activities of olorofim against Coccidioides species. In vitro activity was assessed against 59 clinical Coccidioides isolates. Central nervous system infections were established in mice via intracranial inoculation with Coccidioides immitis arthroconidia. Oral therapy began 48 h postinoculation and consisted of vehicle control, olorofim daily doses of 20 mg/kg (6.67 mg/kg three times daily or 10 mg/kg twice daily) or 40 mg/kg (13.3 mg/kg three times daily or 20 mg/kg twice daily), or fluconazole (25 mg/kg twice daily). Treatment continued for 7 and 14 days in the fungal burden and survival arms, respectively. Fungal burdens were assessed by CFU counts in brains. Olorofim demonstrated potent in vitro activity (MIC range, ≤0.008 to 0.06 μg/ml). Survival was significantly enhanced in mice treated with olorofim. Reductions in brain tissue fungal burdens were also observed on day 9 in the olorofim-treated groups. Improvements in survival and reductions in fungal burdens also occurred with fluconazole. More frequent dosing of olorofim was associated with enhanced survival and greater reductions in fungal burdens. In the group treated with 13.3 mg/kg olorofim three times daily, fungal burdens remained low on day 30 (15 days after treatment was stopped), with undetectable levels in 7 of 10 mice. In contrast, fungal burdens rebounded in all other groups after therapy stopped. Olorofim was highly active in vitro and in vivo against Coccidioides These results demonstrate that olorofim may have a role in the treatment of coccidioidomycosis., (Copyright © 2018 American Society for Microbiology.)

Coccidioidal meningitis can cause significant morbidity, and lifelong antifungal therapy is often required. VT-1598 is a fungus-specific Cyp51 inhibitor that has potent in vitro activity against Coccidioides species. We evaluated the in vivo efficacy of VT-1598 in murine models of central nervous system coccidioidomycosis caused by C. posadasii and C. immitis Infection was introduced via intracranial inoculation, and therapy began 48 h postinoculation. Oral treatments consisted of vehicle control, VT-1598, and positive controls of fluconazole in the C. immitis study and VT-1161 in the C. posadasii study. Treatment continued for 7 and 14 days in the fungal-burden and survival studies, respectively. Fungal burden was assessed in brain tissue collected 24 to 48 h posttreatment in the fungal-burden studies, on the days the mice succumbed to infection, or at prespecified endpoints in the survival studies. VT-1598 plasma concentrations were also measured in the C. posadasii study. VT-1598 resulted in significant improvements in survival in mice infected with either species. In addition, the fungal burden was significantly reduced in the fungal-burden studies. Plasma concentrations 48 h after dosing stopped remained above the VT-1598 MIC against the C. posadasii isolate, although levels were undetectable in the survival study after a 4-week washout. Whereas fungal burden remained suppressed after a 2-week washout in the C. immitis model, a higher fungal burden was observed in the survival arm of the C. posadasii model. This in vivo efficacy supports human studies to establish the utility of VT-1598 for the treatment of coccidioidomycosis., (Copyright © 2018 American Society for Microbiology.)

Background: Echinocandins are recommended as first-line therapy against Candida glabrata infections, although increased resistance to this class has been reported worldwide and they are currently only available for parenteral administration. SCY-078 is an investigational glucan synthase inhibitor that is orally available., Objectives: To evaluate the in vivo efficacy of SCY-078 in an experimental model of invasive candidiasis due to WT and echinocandin-resistant C. glabrata isolates., Methods: Neutropenic ICR mice were inoculated intravenously with a WT isolate (SCY-078 and caspofungin MICs 0.25 and 0.125 mg/L, respectively) or an echinocandin-resistant isolate (SCY-078 and caspofungin MICs 1 and 0.5 mg/L, respectively). Treatment with placebo, SCY-078 (8, 30 or 40 mg/kg orally every 12 h) or caspofungin (1 mg/kg by intraperitoneal injection once daily) began 24 h later. Kidney fungal burden was measured on day 8 post-inoculation., Results: Significant reductions in kidney fungal burden were observed with 30 mg/kg SCY-078 against both isolates and with the 40 mg/kg dose against the echinocandin-resistant isolate. These results were supported by SCY-078 plasma concentration data at the higher doses, where levels above the MICs for both isolates were observed 12 h after the last oral dose. Reductions in fungal burden were also observed with caspofungin against the WT isolate, but not against the resistant isolate., Conclusions: SCY-078 demonstrated in vivo efficacy against infections caused by both WT and echinocandin-resistant C. glabrata isolates in this experimental model. This orally available glucan synthase inhibitor has potential as a therapy against echinocandin-resistant C. glabrata infections., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Previously, modified release itraconazole in the form of a melt-extruded amorphous solid dispersion based on a pH dependent enteric polymer combined with hydrophilic additives (HME-ITZ), exhibited improved in vitro dissolution properties. These properties agreed with pharmacokinetic results in rats showing high and sustained itraconazole (ITZ) systemic levels. The objective of the present study was to better understand the best choice of rodent model for evaluating the pharmacokinetic and efficacy of this orally administered modified release ITZ dosage form against invasive Aspergillus fumigatus. A mouse model and a guinea pig model were investigated and compared to results previously published. In the mouse model, despite similar levels as previously reported values, plasma and lung levels were variable and fungal burden was not statistically different for placebo controls, HME-ITZ and Sporanox ® (ITZ oral solution). This study demonstrated that the mouse model is a poor choice for studying modified release ITZ dosage forms based on pH dependent enteric polymers due to low fluid volume available for dissolution and low intestinal pH. To the contrary, guinea pig was a suitable model to evaluate modified release ITZ dosage forms. Indeed, a significant decrease in lung fungal burden as a result of high and sustained ITZ tissue levels was measured. Sufficiently high intestinal pH and fluids available for dissolution likely facilitated the dissolution process. Despite high ITZ tissue level, the primary therapeutic agent voriconazole exhibited an even more pronounced decrease in fungal burden due to its reported higher clinical efficacy specifically against Aspergillus fumigatus.

Candida albicans, normally found as a commensal in the gut, is a major human fungal pathogen responsible for both mucosal and systemic infections in a wide variety of immunocompromised individuals, including cancer patients and organ transplant recipients. The gastrointestinal tract represents a major portal of entry for the establishment of disseminated candidiasis in many of these individuals. Here we report the development of a diet-based mouse model for disseminated candidiasis acquired via the gastrointestinal tract. Using this model, as well as an appropriate immunosuppression regimen, we demonstrate that dissemination of C. albicans from the gastrointestinal tract can result in mortality within 30 days postinfection. We also show a significant increase in fungal burden in systemic organs, but not gastrointestinal tract organs, upon immunosuppression. Importantly, we demonstrate that the administration of two widely used antifungals, fluconazole and caspofungin, either pre- or postimmunosuppression, significantly reduces fungal burdens. This model should prove to be of significant value for testing the ability of both established and experimental therapeutics to inhibit C. albicans dissemination from the gastrointestinal tract in an immunocompromised host as well as the subsequent mortality that can result from disseminated candidiasis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We evaluated the efficacy of isavuconazole against cryptococcal meningitis. Treatment with either oral isavuconazole (120 mg/kg and 240 mg/kg twice a day [BID]) or fluconazole as the positive control significantly improved survival in mice infected intracranially with either Cryptococcus neoformans USC1597 or H99 and significantly reduced brain fungal burdens for both isolates. Concentrations of isavuconazole in plasma and brain tissue also demonstrated that the greatest improvements in survival and fungal burden were associated with elevated exposures., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The EuropeanAspergillusPCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated "in vivo" specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and β-d-glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and β-d-glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and β-d-glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and β-d-glucan proved optimal (area under the curve [AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests., (Copyright © 2016 White et al.)

Objectives: Candida species are major causes of invasive mycoses in immunocompetent and immunocompromised hosts. Treatment options are limited in the setting of antifungal resistance and increased rates of echinocandin-resistant Candida glabrata have been reported. The novel arylamidine T-2307 demonstrates potent in vitro antifungal activity against Candida species. Our objective was to evaluate the in vitro and in vivo activity of T-2307 against resistant C. glabrata., Methods: In vitro activity was determined against 42 clinical C. glabrata isolates, including 17 echinocandin-resistant strains. Neutropenic ICR mice were inoculated intravenously with an echinocandin-resistant C. glabrata isolate (T-2307; caspofungin MICs ≤0.008 and 0.5 mg/L, respectively). Therapy with vehicle control, T-2307 (0.75, 1.5, 3 or 6 mg/kg subcutaneously once daily) or caspofungin (1 or 10 mg/kg intraperitoneally once daily) began 1 day post-challenge. Kidneys were collected on day 8 and fungal burden was assessed by counting cfu., Results: T-2307 demonstrated potent in vitro activity against C. glabrata (geometric mean MIC 0.0135 mg/L), which was maintained against echinocandin-resistant isolates (geometric mean MIC 0.0083 mg/L). T-2307 also demonstrated in vivo efficacy in mice infected with echinocandin-resistant C. glabrata. Significant reductions in fungal burden were observed at each dosage level of T-2307 compared with control. Reductions in fungal burden were also observed with high-dose caspofungin., Conclusions: T-2307 demonstrated potent in vitro activity against C. glabrata, including echinocandin-resistant isolates, which translated into in vivo efficacy against invasive candidiasis caused by an echinocandin-resistant C. glabrata strain. These results demonstrate the potential for T-2307 as therapy against echinocandin-resistant Candida., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

ASP9726 is an investigational echinocandin with in vitro activity against Aspergillus species. We evaluated the pharmacokinetics and efficacy of this agent in an established guinea pig model of invasive pulmonary aspergillosis. ASP9726 plasma concentrations were measured in guinea pigs administered either a single dose or multiple doses of this agent at 2.5, 5, and 10 mg/kg of body weight/day by subcutaneous injection. Immunosuppressed guinea pigs were inoculated with A. fumigatus AF293, and ASP9726 (2.5, 5, and 10 mg/kg/day), voriconazole (10 mg/kg by oral gavage twice daily), or caspofungin (3 mg/kg/day by intraperitoneal injection) was administered for 8 days. Changes in fungal burden were measured by enumerating CFU and by quantitative PCR of specimens from within the lungs, as well as by analysis of serum (1 → 3)-β-D-glucan and galactomannan. Lung histopathology was also evaluated. ASP9726 plasma concentrations increased in a dose-proportional manner, and the drug was well tolerated at each dose. Each dose of ASP9726, voriconazole, and caspofungin significantly reduced pulmonary fungal burden as measured by quantitative PCR and by determining (1 → 3)-β-D-glucan and galactomannan levels, but only voriconazole significantly reduced numbers of CFU. ASP9726 at 5 mg/kg also significantly improved survival. Histopathology demonstrated morphological changes in hyphae in animals exposed to ASP9726 and caspofungin, consistent with the activities of the echinocandins. These results suggest that ASP9726 may be efficacious for the treatment of invasive pulmonary aspergillosis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We evaluated the in vitro and in vivo activities of the investigational arylamidine T-2307 against echinocandin-resistant Candida albicans. T-2307 demonstrated potent in vitro activity, and daily subcutaneous doses between 0.75 and 6 mg/kg of body weight significantly improved survival and reduced fungal burden compared to placebo control and caspofungin (10 mg/kg/day) in mice with invasive candidiasis caused by an echinocandin-resistant strain. Thus, T-2307 may have potential use in the treatment of echinocandin-resistant C. albicans infections., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The in vitro and in vivo activity of the inositol acyltransferase inhibitor E1210 was evaluated against echinocandin-resistant Candida albicans. E1210 demonstrated potent in vitro activity, and in mice with invasive candidiasis caused by echinocandin-resistant C. albicans, oral doses of 10 and 40 mg E1210/kg of body weight twice daily significantly improved survival and reduced fungal burden compared to those of controls and mice treated with caspofungin (10 mg/kg/day). These results demonstrate the potential use of E1210 against resistant C. albicans infections., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Improved diagnostics are needed to detect invasive pulmonary aspergillosis, a life-threatening infection caused by the pathogenic fungus Aspergillus fumigatus. We are investigating secreted fungal proteases as novel biomarkers for the diagnosis of this disease. Although the A. fumigatus genome encodes a multitude of secreted proteases, few have been experimentally characterized. Here, we employed an unbiased combinatorial library of internally quenched fluorogenic probes to detect infection-associated proteolysis in the lungs of guinea pigs experimentally infected with A. fumigatus. Comparative protease activity profiling revealed a prolyl endopeptidase activity that is reproducibly induced during infection but is not observed in healthy animals. This proteolytic activity was found in four independent animal experiments involving two A. fumigatus isolates. We synthesized a small, focused fluorogenic probe library to define the substrate specificity of the prolyl endopeptidase substrate motif and to identify optimal Probe sequences. These efforts resulted in the identification of a panel of six individual substrate-based fluorescent probes capable of detecting infection in guinea pigs with high statistical significance (P<0.005 in most cases). Receiver operating characteristic analyses demonstrated that this fluorogenic assay could detect A. fumigatus infection-associated proteolysis with comparable sensitivity and specificity as existing diagnostic procedures, suggesting that further optimization of the methodology may lead to improved diagnostics options for invasive pulmonary aspergillosis.

Interest in lateral-flow devices (LFDs) as potential point-of-care assays for the diagnosis of infectious diseases has increased. Our objective was to evaluate the interlaboratory and interstudy reproducibility and the effects of antifungal therapy on an LFD developed for invasive pulmonary aspergillosis (IPA) detection. An established neutropenic guinea pig model of IPA caused by Aspergillus fumigatus was used. At predetermined time points (1 h and 3, 5, and 7 days postinoculation), blood and bronchoalveolar lavage (BAL) fluid were collected from infected and uninfected animals. In a separate experiment, guinea pigs were treated with posaconazole (10 mg/kg of body weight orally [p.o.] twice a day [BID]), voriconazole (10 mg/kg p.o. BID), liposomal amphotericin B (10 mg/kg intraperitoneally [i.p.] once a day [QD]), or caspofungin (2 mg/kg i.p. QD), and samples were collected on days 7 and 11. Each laboratory independently evaluated the IgG monoclonal antibody-based LFD. Galactomannan and (1 → 3)-β-D-glucan were also measured using commercially available kits. Good interlaboratory agreement was observed with the LFD, as the results for 97% (32/33) of the serum and 78.8% (26/33) of the BAL fluid samples from infected animals were in agreement. Good interstudy agreement was also observed. The serum sensitivity of each surrogate-marker assay was reduced in animals treated with antifungals. In contrast, these markers remained elevated within the BAL fluids of treated animals, which was consistent with the fungal burden and histopathology results. These results demonstrate that the LFD assay is reproducible between different laboratories and studies. However, the sensitivity of this assay and other markers of IPA may be reduced with serum in the presence of antifungal therapy.

Miltefosine is an alkyl phosphocholine with good oral bioavailability and in vitro activity against Cryptococcus species that has gained interest as an additional agent for cryptococcal infections. Our objective was to further evaluate the in vivo efficacy of miltefosine in experimental in vivo models of cryptococcal meningoencephalitis and disseminated cryptococcosis. Mice were infected intracranially or intravenously with either C. neoformans USC1597 or H99. Miltefosine treatment (1.8 to 45 mg/kg of body weight orally once daily) began at either 1 h or 1 day postinoculation. Fluconazole (10 mg/kg orally twice daily) or amphotericin B deoxycholate (3 mg/kg intraperitoneally once daily) served as positive controls. In our standard models, miltefosine did not result in significant improvements in survival or reductions in fungal burden against either C. neoformans isolate. There was a trend toward improved survival with miltefosine at 7.2 mg/kg against disseminated cryptococcosis with the H99 strain but only at a low infecting inoculum. In contrast, both fluconazole and amphotericin B significantly improved survival in mice with cryptococcal meningoencephalitis and disseminated cryptococcosis due to USC1597. Amphotericin B also improved survival against both cryptococcal infections caused by H99. Combination therapy with miltefosine demonstrated neither synergy nor antagonism in both models. These results demonstrate limited efficacy of miltefosine and suggest caution with the potential use of this agent for the treatment of C. neoformans infections.

Objectives: Meningoencephalitis caused by Cryptococcus gattii is associated with significant morbidity and the need for aggressive therapy, and often necessitates neurosurgical intervention. We adapted a previously described murine model of cryptococcal meningoencephalitis due to Cryptococcus neoformans to that caused by C. gattii., Methods: Mice were inoculated intracranially with either C. gattii (genotype VGIIa) or C. neoformans. In virulence studies, different C. gattii infecting inocula were compared with a fixed inoculum of C. neoformans, and differences were assessed by survival, brain tissue fungal burden, serum antigen titres and histopathological changes within brain tissue. For treatment, fluconazole or posaconazole (10 mg/kg orally twice daily) was initiated 24 h post-inoculation., Results: C. gattii was more virulent than C. neoformans, as evident by shorter median survival, earlier histopathological changes and higher serum antigen titres. However, no differences in fungal burden or dissemination to other organs were observed among the various groups. In treatment studies, both fluconazole and posaconazole improved the median survival of mice infected with either species. However, neither regimen improved the percentage of animals surviving to the predetermined study endpoint., Conclusions: These results demonstrate the virulence of C. gattii meningoencephalitis and the potential of this model for the assessment of new treatment strategies.

We developed and assessed the diagnostic value of a novel quantitative nested real-time (QNRT) PCR assay targeting the internal transcribed spacer region of ribosomal DNA (rDNA) in a guinea pig model of invasive pulmonary aspergillosis. Groups of 5 immunosuppressed animals that were infected using an aerosol chamber with Aspergillus fumigatus conidia were humanely terminated 1 h postinoculation and at days 3, 5, 7, and 11 postchallenge, and lung tissue, bronchoalveolar lavage (BAL) fluid, whole blood, and serum samples were collected. The QNRT PCR results obtained with the serum and BAL fluid were compared to those achieved with galactomannan and (1→3)-β-d-glucan assays. High fungal burden levels were detected by QNRT PCR in both lung tissue and BAL fluid in all infected animals at each time point, and the sensitivity of each assay in BAL fluid was 100% by day 3 and remained so through the remainder of the study. The sensitivity of detection of fungi in whole blood and serum samples was significantly lower, and some samples remained negative by all three assays despite the advanced stage of the infection. From these data, we can conclude that this novel QNRT PCR method was highly sensitive for the detection of A. fumigatus from different types of samples in this model. In addition, BAL fluid samples appeared to be the most suitable for the early diagnosis of invasive pulmonary aspergillosis. When testing serum, the use of a combination of available assays may increase the possibility of early detection of this opportunistic mycosis.

We compared the rate and extent of anidulafungin's and fluconazole's activity in neutropenic and non-neutropenic mice with Candida albicans invasive candidiasis. In immunocompetent mice, anidulafungin significantly improved survival vs. controls and fluconazole, and significant reductions in (1→3)-β-D-glucan and fungal burden were observed. In neutropenic animals, the highest doses of anidulafungin (5 mg/kg) and fluconazole (10 mg/kg) also improved survival and reduced fungal burden. However, there were no differences in survival between these antifungals as anidulafungin's activity was attenuated in this model. These results demonstrate that the extent of anidulafungin in vivo efficacy may be dependent on host immune status., (© 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.)

In this study we pursued a diagnostic target in Aspergillus fumigatus (AF) by using qualitative Realtime PCR combined with proprietary DNA primers and a hydrolysis probe specific for this fungal target. Qualitative Realtime PCR is a diagnostic tool that utilizes Realtime PCR technology and detects the presence or absence target specific DNA within a predetermined detection range. Respiratory tissue and fluids from experimentally infected guinea pigs were tested by extracting DNA from the samples which were amplified and detected using AF specific DNA primers and probe. This study included qualitative evaluations of all specimens for the presence of the DNA of AF. The findings in the tissues after AF infection were compared to the numbers of spores in aerosolized samples used to inoculate the animals. Results demonstrated that the specific probe and primer set could detect the presence or absence of AF DNA in the sample. The qualitative detection limit of the assay ranged from 6 × 10(4) copies to 6 copies. Since blood cultures are rarely positive for Aspergillosis, our data indicate that qualitative Realtime PCR, in combination with the appropriate DNA primers and probe can serve as an effective diagnostic tool in the early detection of fungal infections.

Previous in vivo studies have reported caspofungin dose escalation to be effective against Candida glabrata with reduced susceptibility. We hypothesized that higher doses of caspofungin would be effective against invasive candidiasis caused by the more virulent species Candida albicans, including isolates resistant to this echinocandin. Immunocompetent mice were inoculated with one of three C. albicans isolates, including one susceptible and two resistant isolates with different FKS1 hot spot 1 point mutations. Mice received daily caspofungin treatment for 7 days and were then followed off therapy for 2 weeks to assess survival. Kidney tissue and blood were collected, and fungal burden and serum (1 → 3)-β-D-glucan were measured. Significant differences in virulence were observed among the three C. albicans isolates, which translated into differences in responses to caspofungin. The most virulent of the resistant isolates studied (isolate 43001; Fks1p F641S) did not respond to caspofungin doses of up to 10 mg/kg of body weight, as there were no differences in survival (survival range, 0 to 12% with treatment), tissue burden, or (1 → 3)-β-D-glucan concentration compared to those for untreated controls. Higher doses of caspofungin did improve survival against the second resistant isolate (53264; Fks1p S645P) that demonstrated reduced virulence (5 and 10 mg/kg; 80% survival). In contrast, caspofungin doses as low as 1 mg/kg improved survival (85 to 95%) and reduced tissue burden and (1 → 3)-β-D-glucan concentration against the susceptible isolate (ATCC 90028). These data suggest that caspofungin dose escalation for invasive candidiasis may not be consistently effective against resistant C. albicans isolates, and this may be associated with the virulence of the strain.

Invasive fusariosis is a highly aggressive fungal infection associated with high mortality in heavily immunocompromised patients. Although posaconazole is efficacious as salvage therapy against infections caused by Fusarium species, concerns remain regarding this agent in the setting of reduced potency. To evaluate the efficacy of posaconazole as treatment or prophylaxis against invasive fusariosis caused by Fusarium solani, we utilized a neutropenic murine model of disseminated disease. ICR mice were administered escalating doses of posaconazole (6.25, 12.5, 25, or 50 mg/kg of body weight twice daily [BID]) by oral gavage beginning 2 days prior to inoculation in the prophylaxis studies or beginning 12 h after inoculation as treatment. Therapy was continued until day 9 postinoculation, and animals were monitored off therapy until day 15 for survival. Fungal burden was assessed as CFU in the kidneys. A clear dose-response relationship was observed, as the highest dose of posaconazole (50 mg/kg) was the most effective in prolonging survival and reducing tissue fungal burden both as prophylaxis and as treatment. This dose response was associated with high posaconazole serum concentrations as measured by bioassay. However, the extent of efficacy was also dependent on the infecting inoculum, as greater increases in survival and reductions in fungal burden were observed with the lower inocula tested. In this model high dosages of posaconazole were effective as treatment and prophylaxis against disseminated fusariosis caused by F. solani.

We compared a lateral flow device to galactomannan and (1-->3)-beta-D-glucan assays to detect invasive aspergillosis in an established guinea pig model of pulmonary disease. The lateral flow device became positive earlier (day 3) than the (1-->3)-beta-D-glucan and galactomannan assays (day 5), with all samples positive by each assay on day 7.

Targeted airway delivery of antifungals as prophylaxis against invasive aspergillosis may lead to high lung drug concentrations while avoiding toxicities associated with systemically administered agents. We evaluated the effectiveness of aerosolizing the intravenous formulation of voriconazole as prophylaxis against invasive pulmonary aspergillosis caused by Aspergillus fumigatus in an established murine model. Inhaled voriconazole significantly improved survival and limited the extent of invasive disease, as assessed by histopathology, compared to control and amphotericin B treatments.

Background: Aminocandin is an investigational echinocandin with excellent activity against Candida species, including Candida albicans and Candida tropicalis. However, few data are available for this agent versus Candida glabrata. We compared the in vitro potency and in vivo efficacy of aminocandin and caspofungin against clinical isolates of C. glabrata including those with reduced caspofungin susceptibility (MIC > 2 mg/L)., Methods: In vitro activity was assessed using microdilution broth susceptibility testing. Three isolates, one with a low and two with elevated caspofungin MICs, were chosen and mice were infected with C. glabrata followed by a single dose of aminocandin or caspofungin (0.5-100 mg/kg), or daily doses of caspofungin (0.07-14.3 mg/kg) begun 1 day after inoculation. Reduction in fungal burden, assessed in kidney tissue on day 8 post-inoculation, was the marker of antifungal response., Results: Aminocandin was more potent than caspofungin against each isolate with reduced caspofungin susceptibility. Mice infected with the caspofungin-susceptible isolate had significant decreases in tissue burden with low doses of either drug. Higher single doses of aminocandin (> or = 10 mg/kg) were required to reduce fungal burden against the two isolates with elevated caspofungin MICs. Single dose administration of caspofungin was ineffective against one of these isolates, and higher daily doses were required to reduce fungal burden., Conclusions: These studies suggest that aminocandin has the potential for extended interval dosing in the treatment of C. glabrata infections caused by susceptible isolates. However, higher doses may be required against isolates with reduced caspofungin susceptibility.

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 log(10) 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.

Extended interval dosing of the echinocandins has been suggested as a potential strategy to overcome the need for daily intravenous administration. This study evaluated the therapeutic and prophylactic efficacy of single doses of aminocandin, a new echinocandin in preclinical development, in a murine model of invasive candidiasis. For therapy, groups of mice were infected with Candida albicans, followed by a single dose of aminocandin (1-15 mg/kg) or placebo (mannitol 5% w/v) administered 1 day after inoculation. As prophylaxis, mice were given a single dose (5 or 30 mg/kg) of aminocandin, caspofungin, or placebo at increasing intervals between dose and inoculation. In both treatment and prophylaxis studies, survival was assessed at 21 days post-inoculation. The reduction in fungal burden was assessed in kidney tissue on day 8 post-inoculation. For treatment, single doses of aminocandin of >/=2.5 mg/kg prolonged survival significantly. In addition, the two doses evaluated for reductions in fungal burden (5 and 15 mg/kg) revealed fungicidal activity. As prophylaxis, both aminocandin and caspofungin 5 and 30 mg/kg prolonged survival when given 7 days before inoculation. Aminocandin and caspofungin 30 mg/kg were both able to prolong survival when the interval between dose and inoculation was increased to 10 days. When this interval was extended to 14 days, only aminocandin 30 mg/kg prolonged survival and reduced fungal burden. These results demonstrate that single doses of aminocandin are effective as treatment and prophylaxis, and suggest that extended interval dosing may be a useful strategy for treating invasive candidiasis.

Serum (1-->3)-beta-D-glucan concentrations were serially measured in the presence and absence of antifungal therapy in a murine model of invasive pulmonary aspergillosis. Serum (1-->3)-beta-D-glucan was detected early during the course of infection, and reductions in this biomarker were associated with improved survival in animals treated with antifungal agents.

Objectives: The therapeutic efficacy of caspofungin alone and in combination with amphotericin B deoxycholate was evaluated in treatment of murine coccidioidomycosis., Methods: Survival and tissue burdens of the spleens and livers were used as antifungal response markers. In a monotherapy study, caspofungin was injected intraperitoneally at 0.1, 0.2, 0.5, 1 and 5 mg/kg per day on days 2 through 15. Amphotericin B deoxycholate was given at 0.1, 0.2 and 0.5 mg/kg intravenously and 1 and 5 mg/kg intraperitoneally three times per week for 2 weeks. In a combination therapy study, amphotericin B deoxycholate at 0.1 mg/kg was administered intravenously three times per week for 2 weeks, respectively, with and without caspofungin intraperitoneally given at 0.1, 0.5 and 5 mg/kg daily on days 2 through 15 post-infection., Results: The study shows that caspofungin and amphotericin B deoxycholate at > or =0.5 and > or =0.1 mg/kg, respectively, were significant in both prolongation of survival and reduction of the tissue fungal burdens of mice compared with controls. No sterilization of either organ was observed with caspofungin doses. In combination therapy, any combination of caspofungin (0.1, 0.5 and 5 mg/kg) with amphotericin B deoxycholate (0.1 mg/kg) improved the period of survival and significantly reduced spleen and liver counts compared with controls., Conclusions: This study indicates that caspofungin has efficacy against systemic coccidioidomycosis in a murine model given in combination with amphotericin B deoxycholate.

We developed a guinea pig model of cryptococcal meningitis to evaluate antifungal agents. Immunosuppressed animals challenged intracranially with Cryptococcus neoformans responded to fluconazole and voriconazole. Disease was monitored by serial cerebrospinal fluid (CSF) cultures and quantitative organ cultures. Our model produces disseminating central nervous system disease and responds to antifungal therapy.

Objective: Prophylactic strategies against invasive pulmonary aspergillosis are often limited by drug interactions and toxicities. Targeted airway delivery of antifungals to the lungs may avoid these pitfalls. We evaluated the effectiveness of an aerosolized nanostructured formulation of itraconazole produced by spray freezing into liquid (SFL) as prophylaxis against invasive pulmonary aspergillosis caused by A. fumigatus., Methods: Immunocompromised Balb/C mice received either itraconazole by oral gavage (Sporanox Oral Liquid [SOL] 30 mg/kg TID) or by aerosolization (SFL 30 mg/kg via 20 min aerosolizations, or control, BID). Dosing began 2 days prior to pulmonary inoculation with A. fumigatus and continued for 7 days post-inoculation. Changes in lung histopathology were also assessed. In the survival arm, mice were monitored over a 5 day period following discontinuation of therapy and survival was assessed by Kaplan-Meier analysis., Results: SFL survival (35%) was greater compared to control (10%; p=0.03) and SOL (0%; p=0.02). Histopathology demonstrated severe invasive disease involving vessels and small airways in control and SOL animals. SFL animals demonstrated colonization with some invasion predominately of large airways., Conclusions: Prophylactic aerosolization of nanostructured SFL significantly improved survival and limited invasive disease of small airways due to A. fumigatus.

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.

The activities of anidulafungin and caspofungin against Candida glabrata were evaluated. MICs, 50% inhibitory concentrations (IC(50) values), and IC(90) values for anidulafungin were lower than those for caspofungin for 16 of 18 strains tested. Anidulafungin has potent in vitro activity against C. glabrata that is maintained against isolates with elevated caspofungin MICs.

Evaluating new therapeutic agents for invasive aspergillosis requires animal models that are reproducible among different laboratories. We therefore evaluated a murine model of aerosol infection in two independent laboratories and found a high level of both intra- and interlaboratory reproducibility of survival, fungal burden over time, and the efficacy of liposomal amphotericin B.

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 delta cnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the delta cnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the delta cnaA 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 delta cnaA 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.

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 DeltagliP 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 DeltagliP 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 DeltagliP 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.

Aerosolized evaporative precipitation into aqueous solution and spray freezing into liquid nanostructured formulations of itraconazole as prophylaxis significantly improved survival relative to commercial itraconazole oral solution and the control in a murine model of invasive pulmonary aspergillosis. Aerosolized administration of nanostructured formulations also achieved high lung tissue concentrations while limiting systemic exposure.

Paecilomyces lilacinus is a fungal pathogen which is generally resistant to amphotericin B and certain other antifungals and is an uncommon cause of devastating fungal keratitis. In the present studies, we evaluated topical voriconazole as therapy for P. lilacinus keratitis in rabbits. Thirty eyes of 15 rabbits were studied. In five animals, the uninfected left eye was treated twice daily with voriconazole (drug control, uninfected eye). In these same animals, the right eye was infected with P. lilacinus but not treated with voriconazole (infection control eye). By day 5, the infection controls had lesions of >2.4 mm in diameter, with conjunctivitis and severe hypopyon, and were sacrificed. In the other 10 rabbits (voriconazole treatment), the right eyes were infected with P. lilacinus and treated with voriconazole beginning on day 3 after infection. Voriconazole therapy caused lesions to decrease during 8 days of therapy, after which rabbits were sacrificed (11 days postinfection). Hyphal masses were present in the control infected eyes and absent in treated infected eyes. Voriconazole was detected in all tissues of treated eyes. Topical voriconazole is effective treatment for P. lilacinus experimental keratitis, and it penetrates more deeply than the corneal tissue.

Immunosuppressed mice were infected intravenously with conidia of Scedosporium prolificans. Treatment was begun 1 day later with liposomal amphotericin B, caspofungin, or both drugs initiated concurrently. Amphotericin B and caspofungin were each effective, but combined therapy did not appear to offer advantages over liposomal amphotericin B alone.