The effect of acquired triazole resistance on abiotic stress tolerance and virulence in Candida auris micro evolved strains

Recently, C. auris become one of the most prominent members of the genus Candida. Since its occurrence, several C. auris outbreaks have been reported worldwide. These outbreaks were associated with isolates displaying decreased susceptibility towards fluconazole, the first-line agent for prophylaxis. Fluconazole is the most frequently used antifungal drug to treat bloodstream Candida infections. The physiological effects of acquired antifungal resistance was investigated in this species using fluconazole, posaconazole and voriconazole resistant mutant strains generated by the in vitro microevolution method. Alterations in antifungal susceptibility and cross resistance were determined by the microdilution method, utilizing azoles (fluconazole, voriconazole, posaconazole), echinocandins (caspofungin, micafungin, anidulafungin) and a polyene (amphotericin B). Changes in the abiotic stress tolerance was examined by spotting assay, using osmotic stressors, cell wall perturbants and a membrane detergent. To evaluate the impact of the acquired resistance on sterol biosynthesis, ergosterol composition of all generated mutant strains were examined. A potential relationship between virulence and acquired antifungal resistance was also studied both in vitro and in vivo. Phagocytosis of the generated strains by J774.2 mouse macrophage-like cells was measured and analyzed by flow cytometry. In the murine infection model fungal burden of the triazole evolved strains was determined in spleen, kidney, liver and brain and compared to the fungal burden associated with the initial azole susceptible strain. Significant differences in virulence of the initial and the generated strains was observed suggesting a potential connection between the virulence and antifungal susceptibility of the emerging fungal pathogen, C. auris.

Novel antifungal activity of Q-Griffithsin, a broad-spectrum antiviral lectin

Background There is a rising global trend in candida strains with high resistance to fluconazole and other antifungal drugs, hence the need for novel agents. Here, we investigated the anti-Candida activity of Q-Griffithsin (Q-GRFT), a lectin naturally produced by the red-sea algae, Griffithsia spp. Methods To assess in vitro growth inhibitory activity, C. albicans was incubated with Q-GRFT on agar plates and in broth media. We investigated GFP-bound Q-GRFT’s ability to adhere to C. albicans using fluorescence microscopy and fluorescence intensity assessments. To demonstrate in vivogrowth inhibitory activity, CBA/J mice were treated per vaginam with Q-GRFT followed by challenge with C. albicans, and fungal burden determined following vaginal lavage. Results Wild type fluorescently labeled Q-GRFT displayed higher fluorescence than the lectin-binding site deficient variant following incubation with C. albicans. Q-GRFT localized around the fungal cells and bound to α-mannan in the cell wall. Q-GRFT significantly inhibited C. albicans growth in broth and on agar plates, disrupted the integrity of the cell wall, and induced ROS formation. The lectin significantly inhibited the growth of C. glabrata, C. parapsilosis and C. krusei, with modest activity against C. auris CDC388 and C. auris CDC389 strains in vitro. Topical treatment resulted in a lower fungal burden compared to the vehicle control group in vaginal candidiasis. Conclusion Q-GRFT binds to and inhibits C. albicans growth both in vitro and in vivo. Further studies are needed to establish the mechanism of growth inhibition.

984. Diagnostic Utility of Bronchoalveolar Lavage Pneumocystis jirovecii DNA Polymerase Chain Reaction Assay in Patients with Hematological Malignancies and Hematopoietic Cell Transplantation

Background Pneumocystis jirovecii, an ubiquitous fungus, can lead to opportunistic pneumonia (PJP) in patients with hematological malignancies (HM) and hematopoietic cell transplantation (HCT) with mild to severe presentation. Unlike patients with HIV, diagnosis of PJP pneumonia is often challenging in patients with HM/HCT possibly related to lower fungal burden versus atypical presentation. The gold standard for diagnosis of PJP from bronchoalveolar lavage fluid (BALF) is cytology, followed by direct fluorescent antigen (DFA), however, in the context of lower fungal burden, quantitative polymerase chain reaction (PCR) is increasingly used. PCR DNA load cut-off for diagnosis of PJP is not established. The objective of this study is to assess the correlation between three tests (cytology, DFA and PCR) and diagnosis of PJP (colonization, possible, probable or proven infection). Methods In this retrospective study at City of Hope, HM/HCT patients with BALF performed to investigate pneumonia who tested positive for any of the 3 tests were included. The study period is from July 2014 to July 2020. All patients had a clinical and radiographic diagnosis of pneumonia. Results Eighty-five patients were identified to have at least one positive diagnostic test for PJP. Twenty (23.5%) patients had a PCR with less than 84 copies/mL, and colonization was suspected in these patients. Of the remaining 65 patients, 46 had all 3 tests done. Twenty seven (58.7%) patients only had positive PCR ranging from 106 to 588,000 copies/mL with negative DFA and cytology. Twelve (26.1%) patients had either DFA or cytology positive with a positive PCR, and in 6 patients (13%) all 3 tests were positive. All of these 18 patients had clinical presentation and radiographic findings consistent with PJP. Quantitative or qualitative serum beta-D-glucan (BDG) level was available in 28 patients and 17 had a positive test with a level >80 pg/ml. Conclusion PJP PCR is a very sensitive test that can lead to early detection of PJP pneumonia in HM/HCT with lower sensitivity of DFA/cytology unless the fungal burden is high. However, the optimal cut off PCR value associated with disease needs to be clinically validated in our patient population and a concurrent serum BDG level can increase diagnostic yield. Disclosures All Authors: No reported disclosures

124. Establishment of a Post-Influenza Aspergillosis Model in Corticosteroid-Immunosuppressed Mice

Background Post-influenza aspergillosis (PIA) is a feared complication in patients with severe influenza, especially those receiving corticosteroids. However, validated murine models of PIA in a background of corticosteroid immunosuppression are lacking, compounding efforts to better characterize the immunopathology and treatment of this emerging entity. Methods 8-week-old female BALB/c mice were infected with ~5% of the lethal dose of a mouse-adapted influenza A/Hong Kong/1968 (H3N2) strain (flu), delivered by aerosolization, versus control (aerosolized saline). Mice then received two intraperitoneal injections of 10 mg cortisone acetate (CA) or mock injections on days 5 and 8 after flu infection. On day 9, mice were intranasally challenged with 50,000 A. fumigatus AF-293 conidia or mock-infected with saline. Survival was monitored until day 16 and infection severity was scored using the modified murine sepsis score (MSS, 0 = healthy to 3 = moribund). Pulmonary fungal burden was determined by an 18S quantitative PCR assay on day 16 or upon death. 15-16 mice per group were assessed across 3 independent experiments. Results Flu infection alone caused modest early morbidity, followed by full recovery of the mice until day 16. Treatment with CA after flu infection led to 12% mortality and increased morbidity that persisted until day 16 (median MSS = 0.8). Similarly, mice infected with AF after CA treatment had 12% mortality and a median MSS of 0.7. Combination of all 3 challenges (flu, CA, and AF) led to 40% mortality and severe morbidity in surviving mice (median MSS = 2.7). Likewise, prior flu infection of CA-treated, AF-infected mice increased the pulmonary fungal burden from 27k to 80k median conidial equivalents. In contrast, mice not receiving CA treatment showed consistently low morbidity (median day-16 MSS = 0.5) and minimal fungal burden after AF challenge, regardless of prior flu infection. Conclusion We have established a model of PIA in CA-immunosuppressed mice that underscores the detrimental effect of corticosteroid therapy on the outcomes of PIA. In the future, we will employ this model to study the impact of various pharmacological interventions on the natural history of PIA in the background of corticosteroid immunosuppression. Disclosures Dimitrios P. Kontoyiannis, MD, Astellas (Consultant)Cidara Therapeutics (Advisor or Review Panel member)Gilead Sciences (Consultant, Grant/Research Support, Other Financial or Material Support, Honoraria)

991. Blockade of the PD-1/PD-L1 Immune Checkpoint Pathway Improves Mortality, Infection Severity, and Fungal Clearance in an Immunosuppressed Murine Model of Invasive Pulmonary Mucormycosis

Background Emerging experimental evidence suggests that immune checkpoint inhibitors (ICIs) enhance antifungal immunity. In addition, there is anecdotal evidence of potential benefit of adjunct PD-1 pathway blockade in patients with intractable mucormycosis. However, proof-of-concept data in animal models are lacking. Therefore, we compared the efficacy of PD-1 and PD-L1 inhibition in an immunosuppressed murine model of invasive pulmonary mucormycosis (IPM). Methods Female 8-9-week-old BALB/c mice were immunosuppressed with cyclophosphamide (150 mg/kg on days -4 and -1, 100 mg/kg on day +3) and cortisone acetate (300 mg/kg on day -1) and infected intranasally with 50,000 Rhizopus arrhizus spores (clinical isolate Ra-749, day 0). On days 0, +2, +4, and +6, mice received intraperitoneal injections of 250 µg/kg PD-1 or PD-L1 blocking antibodies versus (vs.) 250 µg/kg of the corresponding isotype antibodies (all antibodies from Leinco Technologies). Survival was monitored for 7 days post-infection. Infection severity was scored using the murine sepsis score (MSS, 0 = healthy to 3 = moribund). Fungal burden in lung tissue was determined by an 18S quantitative PCR assay on day +7 or upon death. 20 mice per treatment were assessed in 2 independent experiments. Results Control mice with IPM receiving either of the unspecific isotype antibodies developed severe infection (median MSS on day 7, 2.5-3.0) and had a high 7-day mortality (50-55%). Compared to the corresponding isotype control, PD-L1 inhibition provided a strong therapeutic benefit, significantly improving morbidity (median MSS = 1.0 vs. 2.5, p = 0.002), 7-day mortality (15% vs. 50%, p = 0.02), and fungal burden (3.6k vs. 27.2k spore equivalents/lung, p < 0.001). In contrast, blockade of-PD-1 modestly yet non-significantly reduced infection severity (median MSS = 2.1 vs. 3.0, p = 0.48), 7-day mortality (35% vs, 55%, p = 0.12), and fungal burden (5.6k vs. 40.7k spore equivalents/lung, p = 0.09) compared to isotype control. Conclusion Even without concomitant antifungals, blockade of PD-L1 and to a lesser extent of PD-1 improved mortality, infection severity, and fungal clearance in immunosuppressed mice with IPM. Immune phenotyping studies are in progress to better understand the protective antifungal activity of ICIs in IPM. Disclosures Dimitrios P. Kontoyiannis, MD, Astellas (Consultant)Cidara Therapeutics (Advisor or Review Panel member)Gilead Sciences (Consultant, Grant/Research Support, Other Financial or Material Support, Honoraria)

Neutrophil-suppressive activity over T-cell proliferation and fungal clearance in a murine model of Fonsecaea pedrosoi infection

Neutrophils are essential to control several fungal infections. These cells are commonly known for their pro-inflammatory activities. However, some studies have demonstrated the anti-inflammatory properties of neutrophils during certain infectious diseases, culminating in the inhibition of T cell proliferation. Chromoblastomycosis (CBM) is a deep and progressive mycosis that affects thousands of people worldwide. Although neutrophil infiltrates are observed in the lesion histopathology, the fungus can overtake the immune system response and destroy the host-infected tissue. The present study demonstrated that neutropenic animals had an increase in the IL-6 production in the spleen and liver, followed by a lower fungal burden in these organs up to 14 days of infection. Neutropenic animals also showed a lower F. pedrosoi-specific antibody production 14-days post infection and higher T-cell proliferation in the in vitro experiments after stimulation with F. pedrosoi-purified proteins. Taken together, our results suggest that the presence of regulatory neutrophils in the mouse model of F. pedrosoi infection could act favoring the spread of the fungus and the chronicity of the infection. These findings shed light on the CBM treatment, which might target neutrophil polarization as a new therapy approach to treat CBM lesions.

DectiSomes: Glycan Targeting of Liposomal Drugs Improves the Treatment of Disseminated Candidiasis

Candida albicans causes life-threatening disseminated candidiasis. Individuals at greatest risk have weakened immune systems. An outer cell wall, exopolysaccharide matrix, and biofilm rich in oligoglucans and oligomannans help Candida spp. evade host defenses. Even after antifungal treatment, the one-year mortality rate exceeds 25%. Undoubtedly, there is room to improve drug performance. The mammalian C-type lectin pathogen receptors Dectin-1 and Dectin-2 bind to fungal oligoglucans and oligomannans, respectively. We previously coated amphotericin B-loaded liposomes, AmB-LLs, pegylated analogs of AmBisome, with the ligand binding domains of these two Dectins. DectiSomes, DEC1-AmB-LLs and DEC2-AmB-LLs, showed two distinct patterns of binding to the exopolysaccharide matrix surrounding C. albicans hyphae grown in vitro. Here we showed that DectiSomes were preferentially associated with fungal colonies in the kidneys. In a neutropenic mouse model of candidiasis, DEC1-AmB-LLs and DEC2-AmB-LLs delivering only one dose of 0.2 mg/kg AmB reduced the kidney fungal burden several fold relative to AmB-LLs. DEC1-AmB-LLs and DEC2-AmB-LLs increased the percent of surviving mice 2.5-fold and 8.3-fold, respectively, relative to AmB-LLs. Dectin-2 targeting of anidulafungin loaded liposomes, DEC2-AFG-LLs, and of commercial AmBisome, DEC2-AmBisome, reduced fungal burden in the kidneys several fold over their untargeted counterparts. The data herein suggest that targeting of a variety of antifungal drugs to fungal glycans may achieve lower safer effective doses and improve drug efficacy against a variety of invasive fungal infections.

In vivo synergism of free miltefosine or in alginate-based nanocarrier combined with voriconazole on aspergillosis

Aim: To evaluate the activity of miltefosine (MFS), in its free form or loaded-alginate nanoparticles (MFS-AN), alone or combined with voriconazole (VRC) on Aspergillus fumigatus and Aspergillus flavus. Materials & methods: Broth microdilution assay was used for susceptibility testing of Aspergillus isolates, and the antifungal efficacy was assessed using the aspergillosis model in Galleria mellonella larvae. Results: The in vitro synergistic effect of MFS with VRC was observed only against A. fumigatus, whereas both combined therapies (MFS + VRC and MFS-AN + VRC) showed synergism in reducing the larval mortality rate and fungal burden in the larvae infected by A. fumigatus and A. flavus. Conclusions: MFS and MFS-AN combined with VRC may be an important strategy for improving antifungal therapy against aspergillosis.