Azole Research Articles

Progress in mechanisms and immunological treatment of chronic mucocutaneous candidiasis associat-ed with congenital IL-17 pathway deficiency

Chronic mucocutaneous candidiasis (CMC) is a rare, persistent and recurrent infection affecting skin, nails, and oral and genital mucosae. It is mainly caused by Candida albicans and hard to be cured with routine antifungal therapy. Usually, CMC is a primary immunodeficiency disease and can be divided into two categories. The most common one is CMC disease (CMCD), which defined as Candida infection confined to the surface of the skin and mucous membranes and not complicated by systemic Candida albicans infection or other clinical symptoms. The other category is systemic CMC (SCMC) complicated by infections caused by other pathogens, systemic invasive fungal infections, or other clinical symptoms apart from the symptoms of CMCD. It is currently believed that both CMCD and SCMC are related to immunodeficiency caused by gene mutations related to IL-17 signal pathway. The inhibited Th17 proliferation, decreased secretion of IL-17 or IL-22 cytokine, or increased IL-17 or IL-22 neutralizing antibody induced by the mutations promoted the susceptibility to Candida or other pathogens. In the treatment of CMC, in addition to the traditional antifungal drugs such as azoles, polyenes and echinocandins, biological agents and target gene therapy offer potential new therapeutic strategies. This article reviewed the association between congenital immunodeficiency in the IL-17 signaling pathway and CMC, and the possible immunological therapeutic approaches and new therapeutic targets. Key words: Chronic mucocutaneous candidiasis; Candidia albicans; IL-17

Could Fungicides Lead to Azole Drug Resistance in a Cross-Resistance Manner among Environmental Cryptococcus Strains?

Acquired resistance among fungal strains is a growing concern. The reasons for the emergence of this phenomenon, with great clinical implications, are only partially explained and related to the frequent use of fluconazole prophylaxis or therapeutic regimens in medical practice, and evidence of environmental origin of resistance is warranted. Soil exposed to azole fungicides may select acquired resistant fungal strains and improve the proliferation and maintenance of phenotypes in this environment. So far, the greatest focus on cross-resistance in relation to azoles is with genus Aspergillus. In view of fungicides and drugs present similar triazole chemical structure and mechanisms of action, our results showed that fungicides in agricultural areas may decrease fungal sensibility to azole drugs, becoming a potential source of resistance for invasive human mycoses agents. The abusive usage of fungicide in agriculture could play a role in the therapeutic failure of cryptococcosis.

Avaliação físico-química e biológica de nanoemulsão contendo clotrimazol como alternativa terapêutica para o tratamento de candidíase vulvovaginal

Vulvovaginal candidiasis is a high incidence infection, affecting 75% of adult women. However, microorganisms develop protective mechanisms, so it is important to develop strategies. The aim of the present study was the development of clotrimazole (CTZ) loading nanoemulsion for topical treatment of vulvovaginal candidiasis. The nanoemulsions (NE-CTZ) were prepared by the phase inversion temperature method and characterized in relation to the average hydrodynamic diameter (Zav), polydispersity index (PdI), Zeta potential (Zp), Encapsulation Efficiency (EE), Mid Infrared Spectroscopy (FT-MIR), morphology, cytotoxicity in fibroblast cell line (NHI/3T3) and antifungal activity against susceptible (ATCC 24433) and multidrug resistant (ATCC 10231) strains of Candida albicans. The Zav of nanoemulsions was approximately 260 nm, with PdI characteristic of monodisperse systems (~ 0.3) and Zp was equal to -26 mV. Nanoemulsions provided high CTZ incorporation (EE ~ 100%) and FT-MIR assays could corroborate the encapsulation of this active. Morphological analysis showed droplets similar in size to those found for Zav and with irregular round shape. In the cell viability assay it was found that for all concentrations tested (4.8- 0.15μg/ mL) nanoemulsion showed lower cytotoxic potential when compared to free drug. Regarding antifungal assays, NE-CTZ showed inhibitory activity against strains tested in the concentration range of 4.8-0.6μg/ mL (referring to CTZ). Therefore, the developed formulation presents promising physicochemical and biological characteristics for use in the treatment of vulvovaginal candidiasis, specially in cases of antifungal resistance to other azole drugs.

Synergistic effect of ibuprofen with itraconazole and fluconazole against Cryptococcus neoformans

The present study investigated the association of the non-steroidal anti-inflammatory drug ibuprofen with itraconazole, fluconazole and amphotericin B against Cryptococcus neoformans isolates. The minimal inhibitory concentration (MIC) was found according to M27-A3 protocol and in vitro interactions were evaluated using checkerboard microdilution method. Synergism was demonstrated between azoles and ibuprofen for most isolates. However, no synergistic effects were seen when amphotericin B was combined with ibuprofen. Therefore, our results suggest that ibuprofen presents clinical potential when combined with azole drugs in the treatment of cryptococcosis.

Mechanisms of Developing Antifungal Drug Resistance of Candida spp. in Reccurent Urogenital Candidiasis

Urogenital candidiasis is an infectious disease caused by Candida sp., a long recurrent course of which can reduce human reproductive health. Its frequent recurrence is associated with constant influence of provoking factors, noncompliance with treatment regimens, treatment without determining the sensitivity of individual antifungal drugs, local immunity disorders, and increasing antifungal drug resistance. Major drugs for genital candidiasis treatment are azole drugs, but at present there is a tendency toward growing antifungal drug resistance of Candida spp. This review describes antifungal drug resistance mechanisms that are currently under study: the ability of Candida spp. to form biofilms, changes in lipid composition of the fungal cell wall, and genetic mutations of Candida spp. The bulk of the accumulated material concerns the structure and role of the ERG11 gene encoding the sterol-14α-demethylase enzyme, the efflux pump genes CDR1 and CDR2, and the membrane transporter gene MDR1, which are associated with azole drug resistance. These genes are suggested for laboratory diagnostics as molecular genetic markers of antifungal resistance in order to select effective antimycotic therapy. For this review, we used sources from the Scopus, CyberLeninka, MedLine, and Russian Science Citation Index databases.

Persistence of Clonal Azole-Resistant Isolates of Candida albicans from a Patient with Chronic Mucocutaneous Candidiasis in Colombia.

Purpose:The present article describes retrospectively a case of a patient with chronic mucocutaneous candidiasis (CMC) who presented recurrent Candida albicans infection since he was 6 months old. We obtained 16 isolates recovered during a 4-year period. Our purpose was to determinate the susceptibility, genotyping, and the pathogenicity profile in all the isolates.Methods:Sixteen C. albicans were isolated from a 25-year-old male with several recurrent fungal infections admitted to Hospital. The isolates were recovered during 4 years from a different anatomical origin. We typified them by multilocus sequence typing, also we evaluated susceptibility to fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole, caspofungin, and amphotericin B by microdilution method and we also test the pathogenic capacity in the Galleria mellonella model.Results:Genotyping of all clinical isolates showed the persistence of the same diploid sequence type (DST). Isolates changed their susceptibility profile over time, but there were no significant statistical differences in pathogenicity.Conclusion:Herein, a persistent clonal isolates of C. albicans (DST 918) in a patient with CMC, showed changes in its susceptibility profile after several antifungal treatments acquiring gradual resistance to the azole drugs, which did not affect their pathogenicity.

Antifungal Susceptibility of Clinical Isolates and Artificially Produced Multi-azole-resistant Strains of Cryptococcus neoformans (formerly: Cryptococcus grubii) to Ravuconazole.

Ravuconazole (RVCZ) is a newly available human azole drug in Japan since 2018 and is a broad-spectrum antifungal agent that exhibits excellent activity against Candida albicans and Cryptococcus neoformans (formerly: Cryptococcus grubii). The drug is also highly active against isolates that are resistant to fluconazole (FLCZ). In the present study, the in vitro susceptibility to ravuconazole (RVCZ) of Japanese clinical isolates and multi-azole-resistant strains of C. neoformans was investigated using the Clinical & Laboratory Standards Institute (CLSI) M27-A3 test. The minimum inhibitory concentrations for the 14 clinical isolates and the multi-azole-resistant strains were 0.003125-0.125 mg/L and 0.25-0.5 mg/L for RVCZ, respectively. RVCZ is as effective as ITCZ and VRCZ for treating clinical isolates from cats and humans. Moreover, RVCZ is highly effective against multi-azole-resistant strains that encode a protein with a G344S substitution in ERG11. Consequently, RVCZ has considerable potential for use as a therapeutic agent for multi-azole resistant cryptococcosis.

Impact of azole drugs on energetics, kinetics, and ligand migration pathways of CO photo-dissociation in bacterial flavohemoglobins.

Flavohemoglobins (fHbs) are heme proteins found in prokaryotic and eukaryotic microbes. They are involved in NO detoxification through an NO˙ dioxygenase mechanism. The N-terminal heme globin domain allows for binding of gaseous ligands whereas a C-terminal NADH/FADH binding domain facilitates association of redox cofactors necessary for ligand reduction. The NO˙ dioxygenase function is important in facilitating immune resistance by protecting the cell from nitrosative stress brought about by a host organism; as a result, bacterial flavoHbs have recently been considered as targets for the development of new antibiotics. Here, photoacoustic calorimetry and transient absorption spectroscopy have been used to characterize energetics, structural dynamics, and kinetics of CO migration within bacterial flavoHbs from Ralstonia eutropha (FHP) and Staphylococcus aureus (HMPSa) in the presence and absence of antibiotic azole compounds. In FHP, the ligand photo-release is associated with ΔH = 26.2 ± 7.0 kcal mol−1 and ΔV = 25.0 ± 1.5 mL mol−1 while in HMPSa, ΔH = 34.7 ± 8.0 kcal mol−1 and ΔV = 28.6 ± 17 mL mol−1 were observed, suggesting distinct structural changes associated with ligand escape from FHP and HMPSa. In the presence of ketoconazole, the CO escape leads to a more negative enthalpy change and volume change whereas association of miconazole to FHP or HMPSa does not impact the reaction volume. These data are in agreement with the computational results that propose distinct binding sites for ketoconazole and miconazole on CO bound FHP. Miconazole or ketoconazole binding to either protein has only a negligible impact on the CO association rates, indicating that azole drugs do not impact flavoHbs interactions with gaseous ligands but may inhibit the NOD activity through preventing the electron transfer between FAD and heme cofactors.

In Vitro Ravuconazole Susceptibility of Anthropophilic Dermatophyte Strains Isolated from Japanese Patients.

Ravuconazole (RVCZ) is a new human anti-fungal azole drug available in Japan since 2018 and is a broad-spectrum agent that exhibits excellent activity against dermatophytes. In the present study, the in vitro RVCZ susceptibility of clinical isolates of anthropophilic dermatophytes, including Trichophyton interdigitale strains with either low susceptibility to itraconazole (ITCZ) or resistance to terbinafine (TEBR), was investigated using the Clinical & Laboratory Standards Institute M38-A2 test. The MICs of RCVZ for 20 clinical isolates of T. interdigitale were < 0.03125-0.125 mg/L; for 4 clinical isolates of T. rubrum, < 0.03125-0.0625 mg/L; and for 20 clinical isolates of T. tonsurans, < 0.03125 mg/L. Similarly, the MICs of RCVZ for the T. interdigitale strains with either low susceptibility to ITCZ or resistance to TEBR were also < 0.03125 mg/L. To our knowledge, this is first study to investigate the in vitro RVCZ susceptibility of T. interdigitale strains with either low susceptibility to ITCZ or resistance to TEBR. Our results indicated that RVCZ was the most effective drug against these strains.

The Regulatory Mechanism of Cytochrome P450 Family Proteins under Azole Resistance in Neurospora Crassa

: The azoles are the most widely used antifungal drugs. Their target ERG-11 and its downstream protein ERG-5 play significant roles in resistance to the azoles. Both of them belong to the cytochrome P450 family proteins. Except them, there are many other P450 family proteins, but their contribution to fungal drug-resistance is not clear. In this research, we analyzed the effects of cytochrome P450 family proteins on fungal drug-resistance to azoles using the model filamentous fungus N. crassa as research material. Through the azole susceptibility test to the knockout mutants of 18 P450 family proteins including ERG-5, we found that the other 17 P450 family proteins except ERG-5, have the similar susceptibility to azole drugs to the wild-type. Furthermore, we determined the sensitivity of these knockout mutants to benzoic acid, and screened a sensitive mutant to that drug. The corresponding gene (NCU08128) encodes a phenyl-4-monooxygenase.We further analyzed the possible mechanism of the sensitivity of this gene to benzoic acid. We confirmed the roles of the other cytochrome P450 family proteins except ERG-11 and ERG-5in fungal resistance to azole drugs, using the model filamentous fungus N. crassaas research material. The above researches enriched the drug resistance mechanism of filamentous fungi and provided new targets for the development of novel antifungal drugs.

In vitro algaecid effect of itraconazole and ravuconazole on Prototheca species.

Protothecosis is a rare infection in humans, dogs, and cats, and its causative agent is Prototheca species, which consists of achlorophyllic algae that are ubiquitous in natural environments and hosts intestinal flora. Ravuconazole (RVZ) is a new available human azole drug in Japan since 2018 and broad-spectrum antifungal agent. In the present study, the in vitro susceptibility of clinical and environmental isolates of P. wickerhamii, P. zopfii, and P. blaschkeae to itraconazole (ITZ) voriconazole (VRZ), posaconazole (PCZ), and RVZ. RVZ was more potent than the other azoles against Protheca species and has considerable potential for use as a therapeutic agent for human and animal protothecosis.

Regulatory Mechanism of the Atypical AP-1-Like Transcription Factor Yap1 in Cryptococcus neoformans.

AP-1-like transcription factors play evolutionarily conserved roles as redox sensors in eukaryotic oxidative stress responses. In this study, we aimed to elucidate the regulatory mechanism of an atypical yeast AP-1-like protein, Yap1, in the stress response and virulence of Cryptococcus neoformansYAP1 expression was induced and involved not only by oxidative stresses, such as H2O2 and diamide, but also by other environmental stresses, such as osmotic and membrane-destabilizing stresses. Yap1 was distributed throughout both the cytoplasm and the nucleus under basal conditions and more enriched within the nucleus in response to diamide but not to other stresses. Deletion of the C-terminal cysteine-rich domain (c-CRD), where the nuclear export signal resides, increased nuclear enrichment of Yap1 under basal conditions and altered resistance to oxidative stresses but did not affect the role of Yap1 in other stress responses and cellular functions. As a potential upstream regulator of Yap1, we discovered that Mpk1 is positively involved, but Hog1 is mostly dispensable. Pleiotropic roles for Yap1 in diverse biological processes were supported by transcriptome data showing that 162 genes are differentially regulated by Yap1, with further analysis revealing that Yap1 promotes cellular resistance to toxic cellular metabolites produced during glycolysis, such as methylglyoxal. Finally, we demonstrated that Yap1 plays a minor role in the survival of C. neoformans within hosts.IMPORTANCE The human meningitis fungal pathogen, Cryptococcus neoformans, contains the atypical yeast AP-1-like protein Yap1. Yap1 lacks an N-terminal cysteine-rich domain (n-CRD), which is present in other fungal Yap1 orthologs, but has a C-terminal cysteine-rich domain (c-CRD). However, the role of c-CRD and its regulatory mechanism remain unknown. Here, we report that Yap1 is transcriptionally regulated in response to oxidative, osmotic, and membrane-destabilizing stresses partly in an Mpk1-dependent manner, supporting its role in stress resistance. The c-CRD domain contributed to the role of Yap1 only in resistance to certain oxidative stresses and azole drugs but not in other cellular functions. Yap1 has a minor role in the survival of C. neoformans in a murine model of systemic cryptococcosis.

RECENT INVESTIGATIONS AND APPLICATIONS OF AZIDOAZOMETHINE-TETRAZOLE TAUTOMERIC EQUILIBRIUM

Recent investigations of azidoazomethine-tetrazole (azide-tetrazole) tautomeric equilibrium reported from 2014 to 2019 are summarized. Pyridine, pyrimidine, triazine, azole derivatives and their annulated congeners – purines, quinolines, quinazolines are described.

Validation of Human Sterol 14α-Demethylase (CYP51) Druggability: Structure-Guided Design, Synthesis, and Evaluation of Stoichiometric, Functionally Irreversible Inhibitors.

Sterol 14α-demethylases (CYP51) are the cytochrome P450 enzymes required for biosynthesis of sterols in eukaryotes, the major targets for antifungal agents and prospective targets for treatment of protozoan infections. Human CYP51 could be and, for a while, was considered as a potential target for cholesterol-lowering drugs (the role that is now played by statins, which are also in clinical trials for cancer) but revealed high intrinsic resistance to inhibition. While microbial CYP51 enzymes are often inhibited stoichiometrically and functionally irreversibly, no strong inhibitors have been identified for human CYP51. In this study, we used comparative structure/functional analysis of CYP51 orthologs from different biological kingdoms and employed site-directed mutagenesis to elucidate the molecular basis for the resistance of the human enzyme to inhibition and also designed, synthesized, and characterized new compounds. Two of them inhibit human CYP51 functionally irreversibly with their potency approaching the potencies of azole drugs currently used to inhibit microbial CYP51.

Mutations in the anti-sigma H factor RshA confer resistance to econazole and clotrimazole in Mycobacterium smegmatis.

Azole drugs such as econazole, are active on Mycobacterium tuberculosis and Mycobacterium smegmatis ; however, the identification of their target(s) is still pending. It has been reported that mutations in the non-essential system mmpL5-mmpS5 conferred resistance to econazole in M. tuberculosis . We herein report that an azole-resistant mutant screen in M. smegmatis rendered mutations in rshA, encoding a non-essential anti-sigma H protein.

Clinical efficacy and safety of the anti-fungus treatment in advanced elderly patients with fungemia admitted into geriatric ICU

Objective To observe the species distribution, clinical features, efficacy and safety of anti-fungus therapy in advanced elderly patients with fungemia. Methods Clinical data of patients aged 70 years and over with fungemia admitted into geriatric intensive care unit (GICU) of our hospital from Nov. 2012 to Nov. 2017 were retrospectively analyzed. The specie distribution, liver toxicity, differences in biochemical liver and renal functions before and after 28 days of treatment between the caspofungin group and the azole group (fluconazole plus voriconazole), and 28-d survival rate and its risk factors for death were analyzed. Results A total of 72 patients were enrolled, with a median age of 85.5 years (83, 90), a median score of Acute Physiology and Chronic Health Enquiry (APACHE-Ⅱ) of 25.5 (20.3, 31.5), a median score of Sequential Organ Failure Assessment (SOFA) 7 (4.0, 9.8). There were 33 patients (45.8%) with diabetes, 2 patients (2.8%) with hematological diseases, 44 patients (61.1%) with solid tumors and 18 patients (25.0%) with renal insufficiency. Thirty patients (41.7%) needed mechanical ventilation. The detection rate of Candida parapsilosis was 73.6% (53 cases), Candida famata 9.7% (7 cases), Candida tropicalis 5.6% (4 cases), Candida albicans 2.8% (2 cases), Candida glabrata 2.8% (2 cases) and others 5.6% (4 cases). The incidence rate of total liver toxicity was 23.6% after anti-fungus treatment. After 28 days of treatment, 29 patients survived in the caspofungin group (n=42) and 16 patients survived in the azole group (n=30). There were no significant differences in liver and renal function between the two groups before and after treatment. Logistic regression analysis showed that solid tumors (OR: 19.904, 95%CI: 1.944-203.808) and the median APACHE Ⅱ score were the independent risk factors for 28-day death in advanced patients with fungemia. Conclusions Fungemia is becoming more and more prominent in the GICU, which requires clinician’s constant attention in order to provide more basis for the treatment of fungemia in elderly patients. Key words: Intensive care; Fungemia

Rhodotorula spp. in the gut - foe or friend?

Rhodotorula spp. belong to the basidiomyceteous fungi. They are widespread in the environment. Transmission to humans occur mainly through air and food. Intestinal colonization is rather common, but an overgrowth is normally suppressed, since their optimal growth temperature is exceeded in the body. A massive presence in the gut indicates a disturbance of the balance of the microbial flora due to different causes. One particular reason will be the treatment with azoles because this will create an advantage for these azole resistant fungi. First of all, the finding of increased numbers of Rhodotorula in stool specimen is not alarming. In contrast, the colonized human will profit from such a situation since these fungi produce a lot of useful nutrients such as proteins, lipids, folate, and carotinoids. Furthermore, a probiotic effect due to regulation of multiplication of pathogenic bacteria and by neutralizing or destroying their toxins can be anticipated. On the other hand, their massive presence may increase the risk of fungemia and ensuing organ infections especially when the host defense system is hampered. Indeed, Rhodotorula spp. range among the emerging fungal pathogens in the compromised host. However, it can be doubted whether all these opportunistic infections reported originate primarily from the gut.

High prevalence of itraconazole resistance among Candida parapsilosis isolated from Iran.

Background and Purpose:Candida parapsilosis isolates usually have a low minimum inhibitory concentration (MIC) against azoles. Although Candida parapsilosis isolates usually have low MICs against azoles, recent studies candida invasive infections due to azole resistant-C. parapsilosis isolates . Regarding this, the main aim of this study was to determine the susceptibility pattern of Iranian clinical C. parapsilosis against available azole antifungal drugs.Materials and Methods:This study was conducted on 105 previously-identified isolates of C. parapsilosis sensu stricto. For the purpose of the study, the isolates were subjected to antifungal susceptibility testing against fluconazole (FLZ), itraconazole (ITZ), voriconazole (VRZ), and two new azole drugs, namely luliconazole (LUZU) and lanoconazole (LZN). The broth microdilution reference method adopted in this study was according to the Clinical & Laboratory Standards Institute M27-A3 and M27-S4 documents.Results:According to the results, 89% (n=94) of C. parapsilosis isolates showed a MIC of ≥ 1 µg/ml, indicating resistance against ITZ. Multi-azole resistance was observed in 3.8% of the isolates. In addition, LUZU and LZN demonstrated the highest efficacy with the MIC50 values of 0.5 and 1 µg/ml, respectively.Conclusion:The majority of the isolates showed high MIC values against ITZ. This may have been associated with the long-term ITZ prophylaxis/therapy in patients infected with candidiasis. Hence, the adoption of an appropriate antifungal agent is a crucial step for starting the treatment.

Evaluation of the Antifungal Activity of Mentha x piperita (Lamiaceae) of Pancalieri (Turin, Italy) Essential Oil and Its Synergistic Interaction with Azoles.

The promising antimicrobial activity of essential oils (EOs) has led researchers to use them in combination with antimicrobial drugs in order to reduce drug toxicity, side effects, and resistance to single agents. Mentha x piperita, known worldwide as “Mentha of Pancalieri”, is produced locally at Pancalieri (Turin, Italy). The EO from this Mentha species is considered as one of the best mint EOs in the world. In our research, we assessed the antifungal activity of “Mentha of Pancalieri” EO, either alone or in combination with azole drugs (fluconazole, itraconazole, ketoconazole) against a wide panel of yeast and dermatophyte clinical isolates. The EO was analyzed by GC-MS, and its antifungal properties were evaluated by minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) parameters, in accordance with the CLSI guidelines, with some modifications. The interaction of EO with azoles was evaluated through the chequerboard and isobologram methods. The results suggest that this EO exerts a fungicidal activity against yeasts and a fungistatic activity against dermatophytes. Interaction studies with azoles indicated mainly synergistic profiles between itraconazole and EO vs. Candida spp., Cryptococcus neoformans, and Trichophyton mentagrophytes. Thus, the “Mentha of Pancalieri” EO may act as a potential antifungal agent and could serve as a natural adjuvant for fungal infection treatment.

Synergistic Inhibition of Mycotoxigenic Fungi and Mycotoxin Production by Combination of Pomegranate Peel Extract and Azole Fungicide.

Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some Fusarium and Aspergillus spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi Aspergillus flavus and Fusarium proliferatum, both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by A. flavus up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.