A preliminary study of the plasma concentrations of orally administered fluconazole in alpacas (Vicugna pacos)

Alpacas residing in the region endemic for Coccidioides spp. are susceptible to serious, disseminated coccidioidomycosis that may result in death. There is currently no oral antifungal dose recommendation for this species. We used a steady-state study design to assess the pharmacokinetics of oral generic fluconazole in alpacas dosed q 24 h for 14 days. Cohorts of 2–3 animals received fluconazole from 6 to 15 mg/kg/day, and pharmacokinetic analysis was performed after each group of animals in order to make dose adjustments for the next group. The final three animals were used as confirmation of our dose recommendation. The median Tmax was 7 h, and the median Cmax was 1.25 µg/ml per mg/kg dose. The mean dose-normalized 24-h AUC was 41.7 µg h/ml per mg/kg dose (CV = 72%). Based on these results, we recommend alpacas receive a starting dose of oral fluconazole at 10–15 mg/kg/day based on the fluconazole AUC in humans (313–625 µg h/ml). Testing to ascertain putative therapeutic plasma concentrations and monitoring of serum transaminases should be performed.12 month embargo; first published: 24 June 2021This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Posaconazole treatment of refractory coccidioidomycosis in dogs

Background: The majority of dogs with coccidioidomycosis recover with administration of fluconazole or itraconazole, although some cases are refractory or the dogs do not tolerate administration of these medications. Objectives: The objective was to describe the treatment outcomes and therapeutic monitoring of 8 dogs with refractory coccidioidomycosis treated with posaconazole. Animals: Eight dogs with refractory coccidioidomycosis. Methods: Retrospective case series. Medical records from Veterinary Specialty Center of Tucson were searched to identify dogs with refractory coccidioidomycosis that were treated with posaconazole. Clinical information and the results of monitoring trough serum posaconazole concentrations were retrieved. Results: Eight dogs with refractory coccidioidomycosis were treated with 2.5 to 10 mg/kg per day of posaconazole. Six of 8 dogs recovered or developed clinical remission while administered posaconazole. Thirteen serum concentrations from 8 dogs tested were >1 μg/mL (range, 1.52 to >6 μg/mL) and the drug was well-tolerated by 7 dogs. One dog required dosage reductions and treatment was ultimately discontinued because of hepatotoxicosis. Conclusions and Clinical Importance: Posaconazole should be considered as a treatment option for dogs with refractory coccidioidomycosis. Monitoring of indicators of liver function or injury along with therapeutic drug monitoring is recommended to tailor dosage in the event of hepatic toxicosis. © 2021 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC. on behalf of the American College of Veterinary Internal Medicine.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

TNFα Blockade Inhibits Both Initial and Continued Control of Pulmonary Coccidioides

Tumor necrosis factor alpha (TNFα) is a pluripotent cytokine that is important in many infections, though its role in Coccidioides infection remains poorly understood. The need to understand TNFα in Coccidioides infection has increased recently with the widespread use of TNFα inhibitors for a wide variety of autoimmune conditions. Here, we couple the newly developed Coccidioides infection model using strain Cp1038 and C57BL/6 × DBA/2J F1 (B6D2F1) mice. B6D2F1 mice develop long-lasting control of Cp1038. Treatment of B6D2F1 mice with anti-TNFα antibodies permits significant fungal proliferation and death. Additionally, we show that antibody treatment limited to the first 2 weeks of infection was sufficient to induce this same loss of fungal control. Importantly, anti-TNFα antibody treatment initiated after fungal control leads to a loss of host control. These results highlight the importance of TNFα in both the initial control of murine Coccidioides and ongoing suppression of the fungal disease. Copyright © 2022 Powell, Shubitz, Butkiewicz, Trinh, Donovan, Frelinger and Galgiani.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The WOPR family protein Ryp1 is a key regulator of gene expression, development, and virulence in the thermally dimorphic fungal pathogen Coccidioides posadasii

Coccidioides spp. are mammalian fungal pathogens endemic to the Southwestern US and other desert regions of Mexico, Central and South America, with the bulk of US infections occurring in California and Arizona. In the soil, Coccidioides grows in a hyphal form that differentiates into 3-5 micron asexual spores (arthroconidia). When arthroconidia are inhaled by mammals they undergo a unique developmental transition from polar hyphal growth to isotropic expansion with multiple rounds of nuclear division, prior to segmentation, forming large spherules filled with endospores. Very little is understood about the molecular basis of spherule formation. Here we characterize the role of the conserved transcription factor Ryp1 in Coccidioides development. We show that Coccidioides Δryp1 mutants have altered colony morphology under hypha-promoting conditions and are unable to form mature spherules under spherule-promoting conditions. We analyze the transcriptional profile of wildtype and Δryp1 mutant cells under hypha- and spherule-promoting conditions, thereby defining a set of hypha- or spherule-enriched transcripts ("morphology-regulated"genes) that are dependent on Ryp1 for their expression. Forty percent of morphology-regulated expression is Ryp1-dependent, indicating that Ryp1 plays a dual role in both hyphal and spherule development. Ryp1-dependent transcripts include key virulence factors such as SOWgp, which encodes the spherule outer wall glycoprotein. Concordant with its role in spherule development, we find that the Δryp1 mutant is completely avirulent in the mouse model of coccidioidomycosis, indicating that Ryp1-dependent pathways are essential for the ability of Coccidioides to cause disease. Vaccination of C57BL/6 mice with live Δryp1 spores does not provide any protection from lethal C. posadasii intranasal infection, consistent with our findings that the Δryp1 mutant fails to make mature spherules and likely does not express key antigens required for effective vaccination. Taken together, this work identifies the first transcription factor that drives mature spherulation and virulence in Coccidioides. © 2022 Mandel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Vaccine Protection of Mice With Primary Immunodeficiencies Against Disseminated Coccidioidomycosis

Disseminated coccidioidomycosis (DCM), often a severe and refractory disease leading to poor outcomes, is a risk for people with certain primary immunodeficiencies (PID). Several DCM-associated PID (STAT4, STAT3, IFNγ, and Dectin-1) are modeled in mice. To determine if vaccination could provide these mice protection, mice with mutations in Stat4, Stat3, Ifngr1, Clec7a (Dectin-1), and Rag-1 (T- and B-cell deficient) knockout (KO) mice were vaccinated with the live, avirulent, Δcps1 vaccine strain and subsequently challenged intranasally with pathogenic Coccidioides posadasii Silveira strain. Two weeks post-infection, vaccinated mice of all strains except Rag-1 KO had significantly reduced lung and spleen fungal burdens (p<0.05) compared to unvaccinated control mice. Splenic dissemination was prevented in most vaccinated immunodeficient mice while all unvaccinated B6 mice and the Rag-1 KO mice displayed disseminated disease. The mitigation of DCM by Δcps1 vaccination in these mice suggests that it could also benefit humans with immunogenetic risks of severe disease. Copyright © 2022 Powell, Hsu, Butkiewicz, Trinh, Frelinger, Holland, Galgiani and Shubitz.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Reactivation of Coccidioidomycosis in a Mouse Model of Asymptomatic Controlled Disease

The majority of human coccidioidomycosis infections are asymptomatic or self-limited but may have sequestered spherules in highly structured granulomas. Under immunosuppression, reactivation of fungal growth can result in severe disease. B6D2F1 mice asymptomatically infected with C. posadasii strain 1038 were immunosuppressed with dexamethasone (DXM) in drinking water. Treated mice died 16–25 days later, while untreated mice survived (p &lt; 0.001). Flow cytometry of lung granulomas on days 5, 10, 15, and 20 of DXM treatment showed immune cell populations decreased 0.5–1 log compared with untreated mice though neutrophils and CD19+IgD−IgM− cells rebounded by day 20. Histopathology demonstrated loss of granuloma structure by day 5 and increasing spherules through day 20. On day 20, T-cells were nearly absent and disorganized pyogranulomatous lesions included sheets of plasma cells and innumerable spherules. Mice given DXM for 14 days then stopped (DXM stop) survived 6 weeks (9/10). Lung fungal burdens were significantly lower (p = 0.0447) than mice that continued treatment (DXM cont) but higher than untreated mice. Histopathologically, DXM stop mice did not redevelop controlled granulomas by sacrifice, though T-cells were densely scattered throughout the lesions. This demonstrates a mouse model suitable for further study to understand the immunologic components responsible for maintenance control of coccidioidomycosis. © 2022 by the authors.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Evaluation of Host Constitutive and Ex Vivo Coccidioidal Antigen-Stimulated Immune Response in Dogs with Naturally Acquired Coccidioidomycosis

The early innate immune response to coccidioidomycosis has proven to be pivotal in directing the adaptive immune response and disease outcome in mice and humans but is unexplored in dogs. The objectives of this study were to evaluate the innate immune profile of dogs with coccidioidomycosis and determine if differences exist based on the extent of infection (i.e., pulmonary or disseminated). A total of 28 dogs with coccidioidomycosis (pulmonary, n = 16; disseminated, n = 12) and 10 seronegative healthy controls were enrolled. Immunologic testing was performed immediately, without ex vivo incubation (i.e., constitutive), and after coccidioidal antigen stimulation of whole blood cultures. Whole blood cultures were incubated with a phosphate-buffered solution (PBS) (negative control) or a coccidioidal antigen (rCTS1 (105–310); 10 µg/mL) for 24 h. A validated canine-specific multiplex bead-based assay was used to measure 12 cytokines in plasma and cell culture supernatant. Serum C-reactive protein (CRP) was measured with an ELISA assay. Leukocyte expression of toll-like receptors (TLRs)2 and TLR4 was measured using flow cytometry. Dogs with coccidioidomycosis had higher constitutive plasma keratinocyte chemotactic (KC)-like concentrations (p = 0.02) and serum CRP concentrations compared to controls (p < 0.001). Moreover, dogs with pulmonary coccidioidomycosis had higher serum CRP concentrations than those with dissemination (p = 0.001). Peripheral blood leukocytes from dogs with coccidioidomycosis produced higher concentrations of tumor necrosis factor (TNF)-α (p = 0.0003), interleukin (IL)-6 (p = 0.04), interferon (IFN)-γ (p = 0.03), monocyte chemoattractant protein (MCP)-1 (p = 0.02), IL-10 (p = 0.02), and lower IL-8 (p = 0.003) in supernatants following coccidioidal antigen stimulation when compared to those from control dogs. There was no detectable difference between dogs with pulmonary and disseminated disease. No differences in constitutive or stimulated leukocyte TLR2 and TLR4 expression were found. These results provide information about the constitutive and coccidioidal antigen-specific stimulated immune profile in dogs with naturally acquired coccidioidomycosis. © 2023 by the authors.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Mouse Model of a Human STAT4 Point Mutation That Predisposes to Disseminated Coccidiomycosis

STAT4 plays a critical role in the generation of both innate and adaptive immune responses. In the absence of STAT4, Th1 responses, critical for resistance to fungal disease, do not occur. Infection with the dimorphic fungus, Coccidioides, is amajor cause of community-acquired pneumonia in the endemic regions of Arizona and California. In some people and often for unknown reasons, coccidioidal infection results in hematogenous dissemination and progressive disease rather than the typical self-limited pneumonia. Members of three generations in a family developed disseminated coccidioidomycosis, prompting genetic investigation. All affected family members had a single heterozygous base change in STAT4, c.1877A>G, causing substitution of glycine for glutamate at AA626 (STAT4E626G/1). A knockinmouse, heterozygous for the substitution, developed more severe experimental coccidioidomycosis than did wild-typemice. Stat4E626G/1 T cells were deficient in production of IFN-γ after anti-CD3/CD28 stimulation. Spleen cells fromStat4E626G mice showed defective responses to IL-12/IL-18 stimulation in vitro. In vivo, early postinfection, mutant Stat4E626G/1 mice failed to produce IFN-γ and related cytokines in the lung and to accumulate activated adaptive immune cells inmediastinal lymph nodes. Therefore, defective early induction of IFN-γ and adaptive responses by STAT4 prevents normal control of coccidioidomycosis in bothmice and humans. © 2022 The Authors.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]