Multidrug resistance (MDR) transporters such as ATP-Binding Cassette (ABC) and Major Facilitator Superfamily proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased MDR transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of Candida drug resistance (CDR) transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 is one of the few examples of a molecule in which relatively small structural modifications significantly reduced susceptibility to multidrug transporter-mediated resistance.

We reported a 31-year-old man who received renal transplantation for more than 2 years. He was admitted to our hospital on 9 March 2022 due to intermittent diarrhea accompanied by leukopenia for more than 1 month. The patient successively developed high fever, cough, anemia, weight loss, gastrointestinal bleeding, and liver function impairment. Computed tomography (CT) revealed a slight inflammation in the lower lobes of both lungs, enlargement of the lymph nodes in the retroperitoneal and the root of mesenteric areas, and hepatosplenomegaly. Talaromyces marneffei was detected by metagenomics next-generation sequencing (mNGS) in blood and bronchoalveolar lavage fluid, and the pathogen was subsequently verified by blood culture. After endoscopic hemostatic therapy and antifungal therapy with voriconazole and amphotericin B cholesteryl sulfate complex, the patient was successfully discharged. Oral voriconazole was given regularly after discharge. Diarrhea, fever, enlargement of the lymph nodes, and endoscopic evidence of erosion may indicate intestinal T. marneffei infection. Although the mortality of T. marneffei infection after renal transplantation is very high, timely and effective antifungal therapy with amphotericin B cholesteryl sulfate complex is still expected to improve its prognosis.

Cryptococcal meningoencephalitis remains a global health threat with limited treatment options. Currently, the most effective treatment regimens are based on a combination therapy of flucytosine with either amphotericin B or fluconazole. Slow but steady progress is being made toward universal access to flucytosine-based therapies. The broadening access to flucytosine combination therapies will be accompanied by the need for microbiological methods that reliably determine strain susceptibility. This is especially true considering that flucytosine susceptibility can vary widely across clinical isolates. Identifying culture conditions that best represent the host environment are likely optimal and may even be required for accurately determining in vivo flucytosine susceptibility. Here, we report that culture conditions incorporating host-like concentrations of carbon dioxide (CO2) potentiated flucytosine susceptibilities across clinical isolates (10 of 11) that exhibited a range of MIC values under ambient growth conditions (2 to 8 μg/mL) by standard Clinical and Laboratory Standards Institute susceptibility testing. CO2 induced a dose-dependent increase in flucytosine susceptibility between 2- and 8-fold over standard conditions. The CO2-dependent increase in flucytosine susceptibility did not correspond to an increase in fluorouracil susceptibility, indicating a central role for flucytosine uptake through the cytosine permease in the presence of host-like CO2 concentrations. Indeed, the expression of the cytosine permease gene (FCY2) was induced 18- to 60-fold in the mouse lung environment. Therefore, the activity of flucytosine is likely to be very dependent upon host environment and may not be well represented by standard in vitro susceptibility testing. IMPORTANCE Cryptococcus neoformans causes life-threatening infections of the brain. The most effective treatment regimens are based on flucytosine-based combination therapy, which has led to increasingly successful broadening of access to flucytosine globally. Wider use of flucytosine-based therapies for cryptococcal infections will require the ability to reliably determine clinical isolate susceptibilities. We showed that host-like carbon dioxide stress affected flucytosine susceptibility, and this likely occurred through flucytosine uptake. We further showed that the gene encoding the permease, FCY2, and that is responsible for flucytosine uptake was strongly induced during cryptococcal infection. Our data provide insights into the distinctions between the activity of flucytosine in the host environment and during in vitro susceptibility testing.

UNASSIGNED: Azole antifungals are the most commonly used antifungals. The high use of azoles for long-term therapy and prophylaxis is prone to cause resistance. Thus, it is necessary to evaluate the antifungal activity against Candida albicans.
UNASSIGNED: Analyzing the comparison of antifungal exposure on the time-kill curve to Candida albicans.
UNASSIGNED: A case-control study was conducted with a posttest control group design. This study used Candida albicans clinical and ATCC isolates exposed to antifungal solutions with 1 ×, 4 ×, and 16 × minimum inhibitory concentrations (MIC). Antibiotics used included fluconazole, itraconazole, and voriconazole. Candida albicans isolates were incubated with MIC, and the number of colonies was counted at 0, 2, 4, 8, 12, 24, and 48 h. The number of colonies that grew every hour of observation was included in the time-kill curve. The data were then analyzed using an ANOVA test with p <0.05.
UNASSIGNED: The antifungals (fluconazole, itraconazole, and voriconazole) showed fungistatic activity against Candida albicans clinical and ATCC isolates. There was a significant comparison between the antifungal group and the control group at 12, 24, and 48 h. The most significant difference between antifungal and control group was found at 24 h where fluconazole had 95% CI = 0.807-2.061 (p <0.001), itraconazole 95% CI = 0.722-1.976 (p <0.001), and voriconazole CI 95% = 0.807-2.062 (p <0.001).
UNASSIGNED: Fluconazole, itraconazole, and voriconazole were effective in inhibiting the growth of Candida albicans. Maximum inhibition in vitro occurs after 12 h of antifungal exposure.

Invasive fungal infections are difficult to treat with limited drug options, mainly because fungi are eukaryotes and share many cellular mechanisms with the human host. Most current antifungal drugs are either fungistatic or highly toxic. Therefore, there is a critical need to identify important fungal specific drug targets for novel antifungal development. Numerous studies have shown the fungal phosphatidylserine (PS) biosynthetic pathway to be a potential target. It is synthesized from CDP-diacylglycerol and serine, and the fungal PS synthesis route is different from that in mammalian cells, in which preexisting phospholipids are utilized to produce PS in a base-exchange reaction. In this study, we utilized a Saccharomyces cerevisiae heterologous expression system to screen for inhibitors of Cryptococcus PS synthase Cho1, a fungi-specific enzyme essential for cell viability. We identified an anticancer compound, bleomycin, as a positive candidate that showed a phospholipid-dependent antifungal effect. Its inhibition on fungal growth can be restored by ethanolamine supplementation. Further exploration of the mechanism of action showed that bleomycin treatment damaged the mitochondrial membrane in yeast cells, leading to increased generation of reactive oxygen species (ROS), whereas supplementation with ethanolamine helped to rescue bleomycin-induced damage. Our results indicate that bleomycin does not specifically inhibit the PS synthase enzyme; however, it may affect phospholipid biosynthesis through disruption of mitochondrial function, namely, the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which helps cells maintain membrane composition and functionality. IMPORTANCE Invasive fungal pathogens cause significant morbidity and mortality, with over 1.5 million deaths annually. Because fungi are eukaryotes that share much of their cellular machinery with the host, our armamentarium of antifungal drugs is highly limited, with only three classes of antifungal drugs available. Drug toxicity and emerging resistance have limited their use. Hence, targeting fungi-specific enzymes that are important for fungal survival, growth, or virulence poses a strategy for novel antifungal development. In this study, we developed a heterologous expression system to screen for chemical compounds with activity against Cryptococcus phosphatidylserine synthase, Cho1, a fungi-specific enzyme that is essential for viability in C. neoformans. We confirmed the feasibility of this screen method and identified a previously unexplored role of the anticancer compound bleomycin in disrupting mitochondrial function and inhibiting phospholipid synthesis.

UNASSIGNED: To summarize the clinical characteristics, treatment and outcomes of transplant recipients infected with Talaromyces marneffei (TM).
UNASSIGNED: A retrospective analysis was performed on 2 patients with Talaromycosis marneffei (TSM) and transplants at the First Affiliated Hospital of Guangxi Medical University, and a systematic literature review was conducted simultaneously.
UNASSIGNED: This article reported two patients after kidney transplantation who developed fever, cough within 3-4 months. Their haemoglobin was decreased. Their chest computed tomography (CT) showed nodules. TM was detected in their blood or bronchoalveolar lavage fluid samples by next-generation sequencing (NGS). After antifungal treatment with voriconazole (VOR), one patient worsened, the other patient died. A total of 21 patients with TSM after transplants were reported in the literature review. Fourteen underwent kidney transplantation, 4 underwent liver transplantation, 2 underwent lung transplantation, and 1 underwent bone marrow transplantation. The median time from initiating the postoperative immunosuppressive therapy to the onset of symptoms or disease changes was 18 (0.5-140) months. Among them, 9 patients developed fever, 7 patients developed cough or expectoration and 4 patients developed dyspnoea. Haemoglobin was decreased in 10 patients. Pulmonary nodules were found in 7 patients. Among the 21 patients, 7 were diagnosed by positive culture, 6 by biopsy, 5 by culture and biopsy. Of the 21 patients, 13 patients improved by antifungal therapy, 8 patients worsened or died. Seven patients who received amphotericin B followed by itraconazole (ITR) therapy all improved. Regarding the use of immunosuppressants in 12 patients, 9 patients had to discontinue or reduce their medications (6 patients improved, 3 patients worsened or died).
UNASSIGNED: Patients with TSM after transplant often have disseminated infections, involving the respiratory, hematopoietic and so on. Fever, cough, decreased haemoglobin and pulmonary nodules often occur approximately 18 months after surgery. The combined applications of culture, biopsy, NGS are helpful for an early diagnosis. Antifungal therapy with amphotericin B followed by itraconazole is recommended, and the dosage of the immunosuppressant should be adjusted timely.

The heat shock proteins (Hsps) act as a molecular chaperone to stabilize client proteins involved in various cell functions in fungi. Hsps are classified into different families such as HSP90, HSP70, HSP60, HSP40, and small HSPs (sHsps). Hsp90, a well-studied member of the Hsp family proteins, plays a role in growth, cell survival, and pathogenicity in fungi. Hsp70 and sHsps are involved in the development, tolerance to stress conditions, and drug resistance in fungi. Hsp60 is a mitochondrial chaperone, and Hsp40 regulates fungal ATPase machinery. In this review, we describe the cell functions, regulation, and the molecular link of the Hsps with the calcineurin-crz1 calcium signaling pathway for their role in cell survival, growth, virulence, and drug resistance in fungi and related organisms.

Objectives/Hypothesis: To perform a systematic review and meta-analysis to compare the efficacy of and complications associated with antifungal drugs and traditional antiseptic medication for the treatment of otomycosis. Data Sources: The PubMed, EMBASE, GeenMedical, Cochrane Library, CBM, CNKI, VIP and other databases were searched from January 1991 to January 2021. Methods: The systematic literature review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Randomized controlled trials (RCTs) and non-randomized studies (case-control, cohort, and case series) were included to assess the topical use of antifungal drugs and traditional antiseptic medication in patients with otomycosis. The research subjects were patients who were clinically diagnosed with otomycosis and whose external auditory canal secretions were positive for fungi. Funnel plots were used to detect bias, and the Q test was used to assess heterogeneity. The random-effects model was used for meta-analysis. The t-test was used to assess significance. Results: Of the 324 non-duplicate studies screened, 16 studies met the criteria for full-text review, and 7 were included in the meta-analysis. Four studies reported recovery conditions (P = 0.01). Six common complications after medication use were compared, and there were no significant differences. The authors further conducted subgroup analysis according to complications. The differences in the rates of ear distension (P = 0.007), earache (P = 0.03) and tinnitus (P = 0.003) were statistically significant. Conclusion: The results of this meta-analysis and literature review showed that antifungal drugs and traditional antiseptic medication were effective in relieving symptoms in patients with otomycosis, and the two treatments were associated with different complications. Otolaryngologists have the option to use one medication or a combination of two drugs on the basis of the condition. Future research in this area should include RCTs with long-term follow-up to guide the development of otomycosis guidelines to overcome some of the weaknesses found in the literature. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/#myprospero.

The checkerboard broth method based on the Clinical and Laboratory Standards Institute M38-A3 document was used in this study to evaluate the in vitro activity of allicin alone and in combination with the antifungal drugs (griseofulvin, fluconazole, itraconazole and terbinafine) against Microsporum canis isolated from patients with tinea capitis. When allicin was used alone, only weak anti-M. canis effects were found. The MIC50, MIC90 and geometric mean (GM) of terbinafine were the lowest among the compounds tested. Synergism was observed for the combinations of allicin with itraconazole and terbinafine. Only indifference was observed for the combinations of allicin with griseofulvin and fluconazole. Our study illustrated the synergism of allicin in combination with itraconazole and terbinafine, which could be a reference for the treatment of tinea capitis due to M. canis.

Echinocandins such as caspofungin are frontline antifungal drugs that compromise β-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by upregulation of chitin synthesis, thereby sustaining cell wall integrity (CWI). When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a subpopulation of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable supra-high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway. This article has an associated First Person interview with the first author of the paper.