Murine Model Of Disseminated Infection Research Articles

Universal Candidacell-surface-specific monoclonal antibodies protect mice against multidrug-resistant Candida aurisbloodstream infections

Abstract Candidaspecies are the leading cause of life-threatening fungal infections in humans, especially among immunocompromised and hospitalized individuals. Of utmost concern is the emerging “superbug” Candida auriswhich has the potential to disseminate into bloodstream infections with up to 60% mortality in susceptible populations. Importantly, C. aurisis the first inherently multidrug-resistant fungal pathogen, which contributes to a delay in effective treatment and increased mortality rates. Consequently, C. aurisis the first fungal pathogen to cause a serious global public health threat. Our lab has identified and isolated a panel of universal monoclonal antibodies (mAbs) specific to Candidacell wall epitopes that share high homology among various human pathogenic Candidaspecies. Using an established murine model of disseminated infection, we showed that passive transfer of mAb C3.1 (anti-β-1,2-mannotriose) significantly extended survival and reduced fungal burdens and inflammation during early infection. We also showed that mAbs C3.1 and 9F2 (anti-phosphoglycerate kinase 1) significantly enhanced phagocytosis of C. aurisyeast by macrophages and inhibited the proliferation of multiple C. aurisclinical isolates in vitro. Since there are currently no approved antifungal vaccines or antibodies and current treatments are inadequate, critically needed antifungal antibodies can provide long-awaited effective therapies against this deadly pathogen. Our future directions include developing humanized mAbs to evaluate their protective efficacy in genetic mouse models of human immunodeficiency. We will then utilize Fc engineering to derive human IgG1 variants for enhanced complement-based effector efficacy. Supported by grants from NIH /NIAID (R21AI168929) and the Department of Defense (PR171482)

Rapid and Low-Cost Culture-Based Method for Diagnosis of Mucormycosis Using a Mouse Model.

Prompt and targeted antifungal treatment has a positive impact on the clinical outcome of mucormycosis; however, current diagnostic tools used in histopathology laboratories often fail to provide rapid results. Rapid culture-based strategies for early diagnosis of Mucorales infections, which may influence treatment decisions, are urgently needed. Herein, we evaluated a microculture assay for the early diagnosis of mucormycosis in an immunocompetent murine model of disseminated infection, by comparing it with traditional diagnostic methods. The assay specificity was assessed using blood (n = 90) and tissue (n = 90) specimens obtained from mice infected with Rhizopus arrhizus using different inoculum sizes [1 × 104, 1 × 105, and 1 × 106 colony forming units (CFUs)/mouse] and blood (n = 15) and tissue specimens (n = 15) from uninfected mice. Surprisingly, 26 of 90 (28.9%) blood samples revealed positive results by microculture, whereas all blood samples were negative when assayed by conventional culture. The overall positive conventional culture rate for the mouse tissue (kidney) samples was 31.1% (28/90). The calculated sensitivity for kidney microculture was 98.8% [95% confidence interval (CI) 96.6–100], with an assay specificity of 100%. Hence, the microculture assay may be useful for rapid culturing and diagnosis of mucormycosis caused by R. arrhizus directly in blood and tissue samples. Hence, this method may allow for the timely administration of an appropriate treatment.

672. Activity of Ibrexafungerp (Formerly SCY-078) Against Candida auris: In vitro, In Vivo, and Clinical Case Studies of Candidemia

Background Candida auris is a growing global threat; a pathogen associated with high mortality (up to 60%), multidrug resistance, the ability to spread from person-to-person and surface-to-person, presenting high risk for outbreaks in healthcare facilities. Ibrexafungerp is a novel IV/oral glucan synthase inhibitor (triterpenoid) antifungal with activity against Candida, Aspergillus, and Pneumocystis spp., in Phase 3 development.Methods In vitro studies tested ibrexafungerp against >100 clinical isolates of C. auris. Other in vitro studies evaluated the effects of ibrexafungerp against C. auris biofilms. In vivo activity against C. auris was evaluated using a disseminated murine model and a cutaneous infection guinea pig model. In humans, an ongoing open-label trial of ibrexafungerp for treatment of patients with infections caused by C. auris (the CARES study) has been initiated in the United States and India.Results In vitro and in vivo studies demonstrated that ibrexafungerp is active against C. auris, including MDR strains. The MIC mode for ibrexafungerp was 1 μg/mL and the MIC50 and MIC90 were 0.5 and 1 μg/mL, respectively. Many echinocandin-resistant C. auris isolates have shown susceptibility to ibrexafungerp. Furthermore, ibrexafungerp has been shown to reduce biofilm thickness. In animal models of C. auris infection, treatment with ibrexafungerp resulted in improved survival and reduced fungal burden in both the murine model of disseminated infection and the guinea pig model of cutaneous infection as compared with untreated controls. In humans, two patients with difficult to treat C. auris candidemias were enrolled in the CARES study and responded positively to oral ibrexafungerp with eradication of the infection.ConclusionThese data demonstrate that ibrexafungerp possess potent in vitro and in vivo activity as well as promising clinical activity. Therefore, continued clinical evaluation of ibrexafungerp as an option to treat C. auris infections is warranted.Disclosures All authors: No reported disclosures.

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi

ABSTRACTProtein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique β-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in Cryptococcus neoformans and to the yeast-to-hyphal transition in Candida albicans. Therefore, we sought to investigate the importance of the α-subunit, RamB, and the β-subunit, Cdc43, of the A. fumigatus GGTase-I complex to hyphal growth and virulence. Deletion of cdc43 resulted in impaired hyphal morphogenesis and thermo-sensitivity, which was exacerbated during growth in rich media. The Δcdc43 mutant also displayed hypersensitivity to cell wall stress agents and to cell wall synthesis inhibitors, suggesting alterations of cell wall biosynthesis or stress signaling. In support of this, analyses of cell wall content revealed decreased amounts of β-glucan in the Δcdc43 strain. Despite strong in vitro phenotypes, the Δcdc43 mutant was fully virulent in two models of murine invasive aspergillosis, similar to the control strain. We further found that a strain expressing the α-subunit gene, ramB, from a tetracycline-inducible promoter was inviable under non-inducing in vitro growth conditions and was virtually avirulent in both mouse models. Lastly, virulence studies using C. albicans strains with tetracycline-repressible RAM2 or CDC43 expression revealed reduced pathogenicity associated with downregulation of either gene in a murine model of disseminated infection. Together, these findings indicate a differential requirement for protein geranylgeranylation for fungal virulence, and further inform the selection of specific prenyltransferases as promising antifungal drug targets for each pathogen.

Efficacy of echinocandins against murine infections by Diutina (Candida) rugosa

Echinocandins are recommended as a first-line therapy for invasive candidiasis. Candida rugosa was recently transferred to the new genus Diutina. We have determined the in vitro killing kinetics of two echinocandins, anidulafungin, and caspofungin and their in vivo efficacy, administering doses of 5 or 10 mg/kg, and 1 or 5 mg/kg, respectively against 2 clinical strains of D. rugosa. Both drugs showed a fungicidal concentration-dependent activity and, in a neutropenic murine model of disseminated infection, were able to reduce tissue burden and to prolong survival of mice. These results suggest that both echinocandins could be useful to treat infections by this fungus when isolates show minimal inhibitory concentrations within the range of susceptibility for both drugs.

Voriconazole minimum inhibitory concentrations are predictive of treatment outcome in experimental murine infections by Candida glabrata

In this study, 27 clinical isolates of Candida glabrata with voriconazole (VRC) minimum inhibitory concentrations (MICs) ranging from ≤0.03μg/mL to 8μg/mL were tested to determine whether in vitro data are predictive of in vivo efficacy. The efficacy of VRC administered at 40mg/kg was assayed in a neutropenic murine model of disseminated infection by C. glabrata. The reduction in fungal tissue burden in the kidneys was used as a marker of treatment efficacy. VRC reduced the fungal tissue burden in mice infected with strains that had MICs below the epidemiological cut-off value (ECV) of 0.25μg/mL. Variable efficacy of VRC was obtained when the MIC equalled the ECV, and VRC was ineffective when the MIC exceeded the ECV. These results suggest that the use of in vitro data could be useful to predict the outcome for infections by this fungus.

Virulence and Resistance to Antifungal Therapies of Scopulariopsis Species.

Scopulariopsisis an emerging opportunistic fungus characterized by its high resistance to antifungal therapies. We have developed a murine model of disseminated infection in immunosuppressed animals by intravenous inoculation ofScopulariopsis brevicaulisandScopulariopsis brumptii, the most clinically relevant species, in order to evaluate their virulence and their responses to conventional antifungal treatments. Survival and tissue burden studies showed thatS. brumptiiwas more virulent thanS. brevicaulis The three drugs tested, liposomal amphotericin B, posaconazole, and voriconazole, prolonged the survival of mice infected withS. brumptii, but none showed efficacy againstS. brevicaulis The different therapies were only able to modestly reduce the fungal burden of infected tissue; however, in general, despite the high serum levels reached, they showed poor efficacy in the treatment of the infection. Unfortunately, the most effective therapy forScopulariopsisinfections remains unresolved.

In Vivo Synergy of Amphotericin B plus Posaconazole in Murine Aspergillosis.

Aspergillus fumigatus is the main mold causing invasive fungal infection that shows high mortality rates. Therapeutic failure and the increase in drug resistance make it necessary to explore alternative treatments for this infection. We have evaluated the efficacy of amphotericin B at 0.8 mg/kg or 0.3 mg/kg of body weight combined with 40 mg/kg of posaconazole against three A. fumigatus isolates in a murine model of disseminated infection. The combination of the polyene and the azole led to a greater increase in survival and a significantly greater reduction in tissue burden than monotherapies.

Combination therapy in the treatment of experimental invasive fungal infection by Sarocladium (Acremonium) kiliense

Since human infections by Sarocladium (Acremonium) kiliense are usually refractory to antifungal therapy, combinations of drugs are an option that needs exploring. The antifungal activities of anidulafungin plus posaconazole or voriconazole and of amphotericin B plus voriconazole were evaluated and their efficacy was tested in a murine model of disseminated infection by three S. kiliense strains. The results showed that although efficacy was strain-dependent, these combinations work better, in general, than monotherapies. The best results were obtained with the combination of anidulafungin plus posaconazole, although the in vitro assay showed indifference or an antagonistic effect.

Therapies against murine Candida guilliermondii infection, relationship between in vitro antifungal pharmacodynamics and outcome

BackgroundCandida guilliermondii has been recognized as an emerging pathogen showing a decreased susceptibility to fluconazole and considerably high echinocandin MICs. AimsEvaluate the in vitro activity of anidulafungin in comparison to amphotericin B and fluconazole against different isolates of C. guilliermondii, and their efficacy in an immunosuppressed murine model of disseminated infection. MethodsThe in vitro susceptibility of four strains against amphotericin B, fluconazole and anidulafungin was performed by using a reference broth microdilution method and time-kill curves. The in vivo efficacy was evaluated by determination of fungal load reduction in kidneys of infected animals receiving deoxycholate AMB at 0,8mg/kg i.v., liposomal amphotericin B at 10mg/kg i.v., fluconazole at 50mg/kg, or anidulafungin at 10mg/kg. ResultsAmphotericin B and anidulafungin showed fungicidal activity, while fluconazole was fungistatic for all the strains. In the murine model, liposomal amphotericin B at 10mg/kg/day was effective in reducing the tissue burden in kidneys of mice infected with any of the tested strains. However, amphotericin B, anidulafungin and fluconazole were only effective against those strains showing low MIC values. ConclusionsLiposomal amphotericin B showed the higher activity and efficacy against the two strains of C. guilliermondii, in contrast to the poor effect of fluconazole and anidulafungin. Further studies with more isolates of C. guilliermondii representing a wider range of MICs should be carried out to assess whether there is any relationship between MIC values and anidulafungin efficacy.

In vitro pharmacodynamics and in vivo efficacy of fluconazole, amphotericin B and caspofungin in a murine infection by Candida lusitaniae

The in vitro activities of fluconazole (FLC), amphotericin B (AmB) and caspofungin (CSP) were evaluated against three isolates of Candida lusitaniae using time–kill curves. AmB showed in vitro fungicidal activity, whilst FLC and CSP exerted mainly strain-dependent fungistatic activity. The in vivo efficacies of the three drugs were evaluated in a murine model of disseminated infection. The doses administered were FLC 50mg/kg/day, AmB 0.8mg/kg/day and CSP 5mg/kg/day. All three drugs were able to reduce the fungal burden in the kidneys of infected mice, with AmB showing the highest efficacy, followed by CSP. At least in this model, FLC, AmB and CSP are good candidates for treating invasive infections by C. lusitaniae.

Modest efficacy of voriconazole against murine infections by Sporothrix schenckii and lack of efficacy against Sporothrix brasiliensis

The efficacy of voriconazole (VRC) was evaluated against two strains of each of the two most common species causing sporotrichosis, Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, using a murine model of disseminated infection. Voriconazole was administered at doses of 20 or 40mgkg(-1) per day by gavage. The drug showed some efficacy, especially at 40mgkg(-1) per day, in prolonging the survival and reducing fungal load in spleen and liver in mice infected with S. schenckii, whereas in animals infected with S. brasiliensis the drug did not work.

Experimental murine acremoniosis: an emerging opportunistic human infection

Acremonium is an emerging fungal pathogen causing severe infections. We evaluated the virulence of three clinically relevant species within the genus, i.e., Acremonium kiliense (currently Sarocladium kiliense), Acremonium sclerotigenum-A. egyptiacum complex and Acremonium implicatum in a murine model of disseminated infection. Both immunocompetent and immunosuppresssed mice were infected with two inocula concentrations (2 × 10(6) and 2 × 10(8) conidia/animal) of two strains of each species. Tissue burden, mortality rate, histopathology and levels of (1→3)-β-D-glucan were used as virulence markers. None of the species of Acremonium tested was able to cause infection in immunocompetent mice. Conversely, severe infections were produced in immunocompromised mice, the spleen being the most affected organ. In general, the virulence of the Acremonium species tested was low, S. kiliense being the most virulent species.

Evaluation of the correlation of caspofungin MICs and treatment outcome in murine infections by wild type strains of Candida parapsilosis

We have evaluated the in vitro activity of caspofungin against 36 wild-type strains of Candida parapsilosis sensu stricto using 3 techniques: broth microdilution, disk diffusion, and the determination of minimal fungicidal concentration (MFC). The first 2 methods showed a good in vitro activity of caspofungin, but the MFCs were ≥2 dilutions above their corresponding MICs. In a murine model of disseminated infection, we evaluated the efficacy of caspofungin at 5 mg/kg against 8 strains of C. parapsilosis representing different degrees of in vitro susceptibility (0.12–1 μg/mL). All the isolates responded to treatment and (1→3)-β-D-glucan levels were reduced in all the cases; however, the study revealed differences among isolates, since caspofungin reduced the tissue burden of mice infected with isolates with MICs ≤0.5 μg/mL but was less effective against those with MICs of 1 μg/mL.

Efficacy of Amphotericin B at Suboptimal Dose Combined with Voriconazole in a Murine Model of Aspergillus fumigatus Infection with Poor In Vivo Response to the Azole

The combination of amphotericin B at a suboptimal dose (0.3 mg/kg) with voriconazole has shown efficacy in prolonging survival and reducing tissue burden in a murine model of disseminated infection by an isolate of Aspergillus fumigatus that had showed a poor in vivo response to the azole. The efficacy of the combined treatment was higher than that obtained with amphotericin B at 0.8 mg/kg.

Evaluation of the In Vitro Activity of Voriconazole As Predictive of In Vivo Outcome in a Murine Aspergillus fumigatus Infection Model

We have evaluated the in vitro activity of voriconazole against 61 strains of Aspergillus fumigatus by using broth microdilution, disk diffusion, and minimal fungicidal concentration procedures. We observed an excellent correlation between the results obtained with the three methods. Five percent of the strains showed MICs greater than or equal to the epidemiological cutoff value (ECV; 1 μg/ml). To assess whether MICs were predictive of in vivo outcome, we tested the efficacy of voriconazole at 25 mg/kg of body weight daily in an immunosuppressed murine model of disseminated infection using 10 strains representing various patterns of susceptibility to the drug as determined by the in vitro study. Voriconazole prolonged survival and reduced fungal load in the kidneys and brain in those mice infected with strains with MICs of ≤0.25 μg/ml, while in mice infected with strains with MICs of 0.5 to 2 μg/ml, the efficacy was varied and strain dependent and in mice infected with the strain with a MIC of 4 μg/ml, the antifungal did not show efficacy. Voriconazole reduced galactomannan antigenemia against practically all strains with a MIC of <4 μg/ml. Our results demonstrate that some relationship exists between voriconazole MICs and in vivo efficacy; however, further studies testing additional strains are needed to better ascertain which MIC values can predict clinical outcome.

Are epidemiologic cut-off values predictors of the in vivo efficacy of azoles in experimental aspergillosis?

We tried to correlate the in vitro activity and the in vivo efficacy of voriconazole (VRC) and posaconazole (POS) against Aspergillus flavus and Aspergillus niger. The in vitro susceptibility of a large set of isolates was determined by broth microdilution and disk diffusion methods, while the in vivo efficacy was assessed in a murine model of disseminated infection. For A. flavus, VRC showed efficacy even for strains with MICs above the epidemiologic cutoff value (ECV) (1 μg/mL). For A. niger, VRC was ineffective against those strains for which the MIC was 1 dilution below the corresponding ECV (2 μg/mL). POS showed efficacy against all the strains of both species included in the study, although isolates with MICs > ECV were not tested.

Experimental murine model of disseminated infection bySaksenaea vasiformis: successful treatment with posaconazole

We have determined the in vitro activity of amphotericin B (AMB) and posaconazole (PSC) against Saksenaea vasiformis using broth microdilution and disk diffusion methods and determined the minimal fungicidal concentration (MFC). PSC was found to have the greatest in vitro activity in all cases and was the most efficacious in prolonging survival and reducing the fungal load in an immunocompetent murine model of disseminated infection caused by four strains of the fungus.

Efficacy of posaconazole in a murine model of disseminated infection caused by Apophysomyces variabilis

We evaluated the in vitro activity of posaconazole and amphotericin B against several clinical strains of the mucoralean fungus Apophysomyces variabilis, and their efficacy in a murine model of disseminated infection caused by that fungus. The in vitro susceptibility of seven strains of A. variabilis to posaconazole and amphotericin B was determined by using a broth microdilution method. The in vivo efficacy of both drugs, posaconazole at 20 mg/kg twice daily orally by gavage and amphotericin B at 0.8 mg/kg once daily intravenously, was evaluated against six of the strains previously tested in vitro using immunocompetent mice. In general, MICs of both drugs were within the range of susceptibility or intermediate susceptibility. Posaconazole and amphotericin B were able to significantly reduce the percentages of positive cultures in the affected tissues. However, in general, posaconazole significantly improved survival (median, 23 days; range, 7-30 days) compared with untreated controls (median, 6 days; range, 4-7 days) and, in some cases, with respect to the animals treated with amphotericin B (median, 15 days; range, 5-30 days). Our results demonstrate the efficacy of posaconazole in the treatment of a disseminated murine infection caused by A. variabilis. However, further clinical studies are required to ascertain the potential use in human infections caused by this fungus.

Combined Therapy of Voriconazole and Anidulafungin in Murine Infections by Aspergillus flavus

The efficacy of the combination of anidulafungin (AFG) at 1mg/kg plus voriconazole (VRC) at 25mg/kg was evaluated in a murine model of disseminated infection by Aspergillus flavus using three isolates previously tested in vitro. All the combinations showed indifferent in vitro interaction with the exception of one, which showed synergy. In general, the combined treatment prolonged the survival and reduced the fungal load in comparison with AFG alone, and only in a few cases, it improved the results of the VRC monotherapy. The combination of the two drugs and VRC alone reduced the galactomannan levels in serum in comparison with the control group.