Clinical epidemiology of pulmonary aspergillosis in hospitalized patients and contribution of Cyp51A, Yap1, and Cdr1B mutations to voriconazole resistance in etiologic Aspergillus species.

Pulmonary aspergillosis is a life-threatening fungal infection with worldwide distribution. In the present study, clinical epidemiology of pulmonary aspergillosis and antifungal susceptibility of etiologic Aspergillus species were evaluated in one-hundred fifty patients with special focus on the frequency of voriconazole resistance. All the cases were confirmed by the clinical pictures, laboratory findings, and isolation of etiologic Aspergillus species which belonged to two major species, i.e., A. flavus and A. fumigatus. Seventeen isolates displayed voriconazole MIC greater than or equal to the epidemiological cutoff value. Expression of cyp51A, Cdr1B, and Yap1 genes was analyzed in voriconazole-intermediate/resistant isolates. In A. flavus, Cyp51A protein sequencing showed the substitutions T335A and D282E. In the Yap1 gene, A78C replacement led to Q26H amino acid substitution that was not reported previously in A. flavus resistant to voriconazole. No mutations associated with voriconazole resistance were found in the three genes of A. fumigatus. The expression of Yap1 was higher than that of two other genes in both A. flavus and A. fumigatus. Overall, voriconazole-resistant strains of both A. fumigatus and A. flavus demonstrated overexpression of Cdr1B, Cyp51A, and Yap1 genes compared to voriconazole-susceptible strains. Although there are still ambiguous points about the mechanisms of azole resistance, our results showed that mutations were not present in majority of resistant and intermediate isolates, while all of these isolates showed overexpression in all three genes studied. As a conclusion, it seems that the main reason of the emergence of mutation in voriconazole-resistant isolates of A. flavus and A. fumigatus is previous or prolonged exposure to azoles.

In Vitro Susceptibilities of Worldwide Isolates of Intrapulmonary Aspergillus Species and Important Candida Species in Sterile Body Sites against Important Antifungals: Data from the Antimicrobial Testing Leadership and Surveillance Program, 2017-2020.

To understand the changes of resistance in clinically commonly encountered fungi, we used the Antimicrobial Testing Leadership and Surveillance (ATLAS) database to explore in vitro antifungal susceptibilities against clinically important isolates of Aspergillus and Candida species (collected from intrapulmonary and sterile body areas, respectively). We applied the CLSI antifungal 2020 and the EUCAST antifungal 2020 guidelines. From 2017 to 2020, isolates of intrapulmonary Aspergillus fumigatus (n = 660), Aspergillus niger (n = 107), Aspergillus flavus (n = 96), Aspergillus terreus (n = 40), and Aspergillus nidulans species complex (n = 26) and sterile site-originated isolates of Candida albicans (n = 1,810), Candida glabrata (n = 894), Candida krusei (n = 120), Candida dubliniensis (n = 107), Candida lusitaniae (n = 82), Candida guilliermondii (n = 28), and Candida auris (n = 7) were enrolled in this study. Using the EUCAST 2020 breakpoints, it was demonstrated that amphotericin B and posaconazole displayed poor in vitro susceptibility rates against A. fumigatus isolates (<50% and 18.9%, respectively). In contrast, isavuconazole and itraconazole showed high in vitro potency against most Aspergillus isolates (>92%). Most intrapulmonary Aspergillus isolates exhibited MICs of ≤0.06 µg/mL to anidulafungin. Furthermore, intrapulmonary A. fumigatus isolates collected from Italy and the United Kingdom exhibited lower in vitro susceptibility to isavuconazole (72.2% and 69%, respectively) than those in the remaining ATLAS participant countries (>85%). Higher isavuconazole MIC90s against C. auris and C. guilliermondii (1 and 4 µg/mL, respectively) were observed compared to the other five Candida species. Despite the aforementioned MICs and susceptibilities against fungi, research needs to consider the pharmacokinetic (PK) profiles, pharmacodynamic (PD) parameters, and clinical treatment experience with antifungals against specific Aspergillus species. IMPORTANCE In addition to monitoring the antifungal susceptibilities of clinically important fungi, reviewing the PK/PD indices and the clinical therapy experience of antifungals under evaluation are important to guide an appropriate antifungal prescription. The efficacies of liposomal amphotericin B complex and anidulafungin for the treatment of pulmonary aspergillosis caused by different Aspergillus species need to be periodically evaluated in the future.

Treatment of otomycosis with clotrimazole: results accordingly with the fungus isolated.

BACKGROUND: Otomycosis is usually caused by Candida spp or Aspergillus spp. While Candida is usually multissensitive to available antifungals, Aspergillus is not. Topical antifungals for otomycosis that are available in Portugal are scarce, and systemic treatments have too many interactions and contraindications. OBJECTIVES: Determine otomycosis epidemiology, microbiology and treatment results. METHODS: Observational study that included patients followed in Professor Doutor Fernando Fonseca Hospital, between 2011 and 2020. Otomycosis diagnosis was obtained through ear drainage culture, and every case was treated with 1% clotrimazole ear drops plus ear cleaning once per week. RESULTS: Aspergillus was found in ear drainage culture in 43.9% of patients and Candida in the remaining. There was a significant statistical difference between patients with otomycosis caused by Aspergillus versus Candida in treatment duration from 25.0 days (16.5-43.0) versus 14.0 days (7.0-18.5) (p < .001), respectively. CONCLUSIONS: Otomycosis was more frequently caused by Candida, and this type of otomycosis is treated faster with clotrimazole 10 mg/dL plus ear cleaning, when compared with otomycosis by Aspergillus. SIGNIFICANCE: If otomycosis causative agent is identified or suspected, a prediction of the time needed till the resolution of otomycosis can be made, when clotrimazole ear drops are used.

Echinocandins - structure, mechanism of action and use in antifungal therapy.

With increasing number of immunocompromised patients as well as drug resistance in fungi, the risk of fatal fungal infections in humans increases as well. The action of echinocandins is based on the inhibition of ß-(1,3)-d-glucan synthesis that builds the fungal cell wall. Caspofungin, micafungin, anidulafungin and rezafungin are semi-synthetic cyclic lipopeptides. Their specific chemical structure possess a potential to obtain novel derivatives with better pharmacological properties resulting in more effective treatment, especially in infections caused by Candida and Aspergillus species. In this review we summarise information about echinocandins with closer look on their chemical structure, mechanism of action, drug resistance and usage in clinical practice. We also introduce actual trends in modification of this antifungals as well as new methods of their administration, and additional use in viral and bacterial infections.

Safety and Effectiveness of Isavuconazole Treatment for Fungal Infections in Solid Organ Transplant Recipients (ISASOT Study).

Isavuconazole (ISA) is an alternative treatment for Aspergillus spp. and other fungal infections, but evidence regarding its use in solid organ transplant recipients (SOTR) is scarce. All SOTR who received ISA for treatment of a fungal infection (FI) at our center from December 2017 to January 2021 were included. The duration of the treatment depended on the type of infection. All patients were followed up to 3 months after treatment. Fifty-three SOTR were included, and the majority (44, 83%) were lung transplant recipients. The most frequently treated FI was tracheobronchitis (25, 46.3%). Aspergillus spp. (43, 81.1%); specially A. flavus (16, 37.2%) and A. fumigatus (12, 27.9%), was the most frequent etiology. Other filamentous fungi including one mucormycosis, and four yeast infections were treated. The median duration of treatment was 81 days (IQR 15-197). Mild gamma-glutamyltransferase elevation was the most frequent adverse event (34%). ISA was prematurely discontinued in six patients (11.3%) due to mild hepatotoxicity (2), fatigue (2), gastrointestinal intolerance (1) and myopathy (1). The mean tacrolimus dose decrease was 30% after starting ISA. Seven patients received ISA with mTOR inhibitors with good tolerability. Two patients developed breakthrough FI (3.8%). Among patients who completed the treatment, 27 (50.9%) showed clinical cure and 15 (34.1%) presented fungal persistence. Three patients (6%) died while on ISA due to FI. ISA was well tolerated and appeared to be an effective treatment for FI in SOTR. IMPORTANCE We describe 53 solid organ transplant recipients treated with isavuconazole for fungal infections. Because its use in clinical practice, there is scarce data of its use in solid organ transplant recipients, where interactions with calcineurin inhibitors and mTOR and adverse drug events have limited the use of other triazoles. To the best of our knowledge, this is the first article describing the safety regarding adverse events and drug interactions of isavuconazole for the treatment of fungal infections in a cohort of solid organ transplant recipients. Also, although this is a noncomparative study, we report some real world effectivity data of these patients, including treatment of non-Aspergillus fungal infections.

A sensitive and rapid bioanalytical method for the quantitative determination of luliconazole in rabbit eye tissues using UPLC-MS/MS assay.

Luliconazole (LCZ) is a novel antifungal imidazole with broad-spectrum and high susceptibility of Aspergillus and Fusarium are the dominant species of fungal keratitis, may potentially be a new medical treatment option for ocular fungal infection. To evaluate LCZ distribution in ocular tissues after topical application for the development of ophthalmic delivery system, it is important to have a bioanalytical method for measuring the drug concentrations in different ocular tissues and aqueous humor (AH). A selective and sensitive ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantification of LCZ in rabbit ocular tissues, including conjunctiva, cornea, AH, iris, lens, vitreous humor (VH), retinal choroid and sclera, using lanoconazole as internal standard (IS). Chromatographic separation was achieved on a Xterra MS, C18 column (2.1 × 50 mm, 3.5 µm) using mobile phase with formic acid solution (0.2%, v/v): acetonitrile (50:50, v/v) at a flow rate of 0.2 ml/min, and the run time was 2.5 min. Detection was performed using the transitions 354.1 → 150.3 m/z for LCZ and 320.1 → 150.3 m/z for IS by positive ion electrospray ionization in multiple reaction monitoring (MRM) mode. Method validation was conducted in accordance with U.S. Food and Drug Administration's regulatory guidelines for bioanalytical method validation. The calibration curves were linear over the concentration range from 2.80 ng/ml to 2038 ng/ml for conjunctiva, cornea and sclera, 2.09 ng/ml to 1019 ng/ml for AH, 2.09 ng/ml to 509.5 ng/ml for iris, 2.09 ng/ml to 203.8 ng/ml for retinal choroid and VH, 2.04 ng/ml to 101.9 ng/ml for lens, with all the squared correlation coefficients (r2) more than 0.99. The accuracy of the method was within the acceptable limit of 89.34%∼112.78% at the lower limit of quantification and other concentrations, Inter-day and intra-day precision values, expressed in terms of RSD (%), in all tissues were within 15% at all concentrations. The mean recoveries of LCZ in rabbit ocular tissues was 84.85%∼100.52%. No interference was found due to matrix components. Luliconazole was stable during the stability studies, including autosampler stability, benchtop stability, freeze/thaw stability and long-term stability. The method was successfully applied to the ocular pharmacokinetic and tissues distribution studies of LCZ in rabbit after topical administration of LCZ ophthalmic drug delivery system.

Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

The accuracy and clinical impact of the morphological identification of Aspergillus species in the age of cryptic species: A single-centre study.

BACKGROUND: Aspergillus spp. is identified morphologically without antifungal susceptibility tests (ASTs) in most clinical laboratories. The aim of this study was to examine the clinical impact of the morphological identification of Aspergillus spp. to ensure the adequate clinical management of Aspergillus infections. PATIENTS/METHODS: Aspergillus isolates (n = 126) from distinct antifungal treatment-naïve patients with aspergillosis were first identified morphologically, followed by species-level identification via DNA sequencing. An AST for itraconazole (ITC) and voriconazole (VRC) was performed on each Aspergillus isolate. RESULTS: Based on the genetic test results, morphology-based identification was accurate for >95% of the isolates at the species sensu lato level although the test concordance of Aspergillus spp. with low detection rates was low. The rates of cryptic species were found to be 1.2% among the isolates of A. fumigatus complex and 96.8% in the A. niger complex. Cryptic species with lower susceptibilities to antifungal drugs than sensu stricto species among the same Aspergillus section were as follows: The A. lentulus (n = 1) isolates had low susceptibilities to azoles among the A. fumigatus complex species (n = 86), and A. tubingensis isolates (n = 18) exhibited lower susceptibility to azoles among the A. niger complex species (n = 31). CONCLUSION: Diagnostic accuracy was high at the A. fumigatus and A. niger complex level. However, in the presence of cryptic species, a solely morphological identification was insufficient. Particularly, ITC and VRC might be inappropriate for aspergillosis treatment when the A. niger complex is identified morphologically because it is possible that the Aspergillus isolate is A. tubingensis.

Epilobium angustifolium L. Essential Oil-Biological Activity and Enhancement of the Skin Penetration of Drugs-In Vitro Study.

Epilobium angustifolium L. is a popular medicinal plant found in many regions of the world. This plant contains small amounts of essential oil whose composition and properties have not been extensively investigated. There are few reports in the literature on the antioxidant and antifungal properties of this essential oil and the possibility of applying it as a potential promoter of the skin penetration of drugs. The essential oil was obtained by distillation using a Clavenger type apparatus. The chemical composition was analyzed by the GC-MS method. The major active compounds of E. angustifolium L. essential oil (EOEa) were terpenes, including α-caryophyllene oxide, eucalyptol, ß-linalool, camphor, (S)-carvone, and ß-caryophyllene. The analyzed essential oil was also characterized by antioxidant activity amounting to 78% RSA (Radical Scavenging Activity). Antifungal activity against the strains Aspergillus niger, A. ochraceus, A. parasiticum, and Penicillium cyclopium was also determined. The largest inhibition zone was observed for strains from the Aspergillus group. The EOEa enhanced the percutaneous penetration of ibuprofen and lidocaine. After a 24 h test, the content of terpene in the skin and the acceptor fluid was examined. It has been shown that the main compounds contained in the essential oil do not penetrate through the skin, but accumulate in it. Additionally, FTIR-ATR analysis showed a disturbance of the stratum corneum (SC) lipids caused by the essential oil application. Due to its rich composition and high biological activity, EOEa may be a potential candidate to be applied, for example, in the pharmaceutical or cosmetic industries. Moreover, due to the reaction of the essential oil components with SC lipids, the EOEa could be an effective permeation enhancer of topically applied hydrophilic and lipophilic drugs.

Synthesis, Biological Evaluation, and Structure-Activity Relationships of 4-Aminopiperidines as Novel Antifungal Agents Targeting Ergosterol Biosynthesis.

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.

Antifungal and antiaflatoxigenic assessment of new Cu(II)-pq complexes against Aspergillus parasiticus, in dark conditions and under visible irradiation.

The issue of food contamination by fungi and aflatoxins; constitutes a serious concern not only for human/animal health but also for agriculture and the economy. Aflatoxins are secondary metabolites produced by certain filamentous fungi and contaminate a variety of foodstuffs. In this context, control of fungal growth and aflatoxin contamination appears to be important. The present study aimed to investigate new Cu(I) and Cu(II)-quinoxaline complexes, namely [Cu(2,2´-pq)(NO3)](NO3) (1), [Cu(2,2´-pq)2(NO3)](NO3)·6H2O (2) and [Cu(2,2΄-pq)2](BF4) (3), where 2,2´-pq is 2-(2'-pyridyl quinoxaline), as antifungal agents against Aspergillus parasiticus. All complexes, the ligand and the starting material Cu(NO3)2-3H2O, regardless of the concentration used, caused inhibition of A. parasiticus growth ranged from 8.52 to 33.33%. The fungal growth inhibition was triggered when irradiation in visible (λ > 400 nm) was continuously applied (range 18.36-57.20%). The highest inhibitory activity was exhibited by the complex [Cu(2,2´-pq)2(NO3)](NO3)·6H2O and for this reason, it was selected to be studied for its ability to suppress aflatoxin B1 produced by A. parasiticus. AFB1 production after the irradiation process was found to be suppressed by 25% compared to AFB1 produced in dark conditions.

In Vitro Antifungal and Antibacterial Effects of Biosynthesized Zinc Oxide Nanoparticles Against Selected Pathogenic and Non-Pathogenic Strains.

We have developed a simple, robust environment-friendly and efficient method for ZnO nanoparticles biosynthesis using Dalbergia sissoo fresh leaf extract. Before using these nanoparticles for antimicrobial assay, a detailed characterization was performed using techniques like Ultraviolet/Visible (UV/Vis) spectroscopy, Particle size analysis (PSA), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM),Transmission electron microscopy (TEM) etc. The average size of biosynthesized ZnO nanoparticles was around 30 nm and they were pure and crystalline by nature. The effectiveness of these biosynthesized nanoparticles were checked against both pathogenic and non-pathogenic microbes. A total of eight bacterial strains-Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Klebsilla pneumoniae, Staphylococcus aureus, Streptococcus entericus, Bacillus cereus, Pantoea cypripedii and three fungal strains-Candida albicans, Aspergilus niger and Aspergilus flavus were studied to have a clear view of the spectrum of ZnO nanoparticles anti-microbial activity. The effectiveness of biosynthesized ZnO nanoparticles against the microbes was found to be better than the standard reference antibiotics used (streptomycin, chloramphenicol and rifampicin). The results seem to be very promising and can be used for some practical applications of ZnO nanoparticles in nearfuture.

Cyclopeptide Derivatives from the Sponge-Derived Fungus Acremonium persicinum F10.

Cyclopeptides usually play a pivotal role, either in the viability or virulence of fungi. Two types of cyclopeptides, six new hydroxamate siderophore cyclohexapeptides (1-6), including acremonpeptides E and F, and their complexes with aluminum and ferric ions; one new cyclic pentapeptolide, aselacin D (9); together with a known compound, aselacin C (10), were isolated and characterized from the sponge-derived fungus Acremonium persicinum F10. In addition, two new siderophore analogues chelating gallium ions (Ga3+), Ga (III)-acremonpeptide E (7) and Ga (III)-acremonpeptide F (8), using isolated acremonpeptides E and F, were prepared. The planar structures of 1-10 were elucidated by HRESIMS and (1D and 2D) NMR. The absolute configurations of amino acids were determined by means of the advanced Marfey's method and X-ray single-crystal diffraction analysis. X-ray fluorescence (XRF) spectrometer was performed to disclose the elements of compound 1, indicating the existence of aluminum (Al). Al (III)-acremonpeptides E (1), Ga (III)-acremonpeptides E (5), Al (III)-acremonpeptide F (7), and Ga (III)-acremonpeptide F (8) displayed high in vitro anti-fungal activities, which are comparable to amphotericin B, against Aspergillus fumigatus and Aspergillus niger.

Antimicrobial, antioxidant and cytotoxic properties of Chenopodium glaucum L.

We evaluated phytochemical composition, antibacterial, antifungal, anti-oxidant and cytotoxic properties of aqueous (water) and organic extracts (methanol, ethyl acetate and n-hexane) of Chenopodium glaucum. Highest phenolic content 45 mg gallic acid equivalents (GAE)/g d.w was found in aqueous extract followed by ethyl acetate (41mg GAE/g d.w) and methanol extract (34.46 mg GAE/g d.w). Antibacterial potential of aqueous and organic extracts of C. glaucum was examined against Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli and Staphylococcus epidermidis. The aqueous, methanolic, ethyl acetate, and n-hexane extract showed antibacterial activity against A. baumannii, K. pneumoniae, E. coli and S. epidermidis. However, against A. baumannii significantly higher inhibition zone (19 mm and 18.96 mm respectively) was shown by ethyl acetate and methanol extracts. Aqueous extract possessed highest growth inhibition (11 mm) against E. coli. Aqueous, ethyl acetate and methanol extracts showed 9 mm, 10 mm, and 10.33 mm zone of inhibition against the K. pneumoniae. For antifungal activity, the extracts were less effective against Aspergillus niger but showed strong antifungal activity against Aspergillus flavus (A. flavus). The antioxidant activity was measured as DPPH (2, 2-diphenyl-1-picrylhydrazyl), H2O2 and ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity of free radicals. All the organic extracts of C. glaucum possessed ABTS, DPPH and H2O2 scavenging properties. The highest cytotoxic activity measured as half maximal inhibitory concentration (IC50) against human lungs carcinoma cells was recorded for methanolic (IC50 = 16 µg/mL) and n-hexane (IC50 = 25 µg/mL) extracts, respectively. The Gas chromatography-mass spectrometry (GC-MS) analysis showed 4 major and 26 minor compounds in n-hexane extract and 4 major and 7 minor compounds in methanol extract of the C. glaucum. It is concluded that aqueous and organic extracts of C. glaucum would be potential therapeutic agents and could be exploited on a pilot scale to treat human pathogenic diseases.

The effects of antifungal therapy on the recurrence of aspergillus infection after pulmonary aspergilloma resection: a study protocol for a single-center, prospective, non-blind, randomized, 24-month, parallel group study.

BACKGROUND: In recent years, the incidence of pulmonary aspergilloma has increased. The harm of aspergilloma is life-threatening massive hemoptysis, and the conventional treatment is surgical treatment. However, whether the antifungal treatment after surgery is required and the course of treatment before and after surgery are still unclear. METHODS: In this study, patients with pulmonary aspergilloma confirmed pathologically after surgery will be selected as subjects to conduct a single-center, randomized, parallel grouping, prospective, 2-year clinical study. Through regular visits, the recurrence of aspergillus infection, quality of life, lung function indicators, safety of antifungal therapy and other indicators were recorded to evaluate the recurrence risk of aspergillus infection and safety of antifungal agents. Cox proportional risk regression model was used to analyze the influencing factors of antifungal therapy on aspergillus infection recurrence after aspergillus bulbectomy. Cox multiple regression model was used for optimal model fitting, and regression coefficient (ß), relative risk (RR) and 95% confidence interval of RR were calculated. DISCUSSION: The study will explore whether antifungal therapy could improve the quality of life, reduce the recurrence of aspergillus infection, and ultimately improve the prognosis of patients with aspergilloma. The study results will provide high-quality evidence-based medical evidence for the formulation, revision and optimization of international and domestic clinical guidelines and expert consensus on chronic aspergillus lung disease, effectively improve the clinical treatment effect of aspergilloma, and form the latest concept of diagnosis and treatment of aspergilloma. TRIAL REGISTRATION: The trial was registered on the Chinese Clinical Trial Registry website ( https://www.chictr.org.cn/showprojen.aspx?proj=33231 ). Registration number: ChiCTR1800019990.

CYP51 Paralogue Structure Is Associated with Intrinsic Azole Resistance in Fungi.

Azoles are the most commonly used clinical antifungal therapy and also play an important role in control of plant pathogens. Intrinsic resistance to the azole class of fungicides, which target lanosterol demethylase (CYP51), is observed in many fungal species; however, the mechanisms underpinning this phenomenon are unknown. In this study, 5 azole-resistant Penicillium isolates from patients attending the UK National Aspergillosis Centre that could not be morphologically identified to species level were analyzed by genome sequencing. The genomes and CYP51 paralogue structure from these isolates were compared with those of 46 representative fungal isolates to identify to species level and examine possible mechanisms of drug resistance. Analysis of CYP51 paralogues showed that azole-resistant isolates from this study (n = 2) and from public databases (n = 6) contained a new CYP51 paralogue, CYP51D, which was associated with azole resistance in 6/8 cases and never occurred in azole-sensitive species (46/46 tested). Furthermore, one isolate from this study and an azole-resistant Aspergillus fumigatiaffinis isolate were shown to encode a CYP51A paralogue, CYP51A2. Introduction of CYP51A2 to the closely related but azole-sensitive Aspergillus fumigatus resulted in azole resistance. The identification of novel CYP51A and CYP51D paralogues in resistant fungi and the observation that resistance to azoles can be conferred by introducing a CYP51A paralogue from a resistant species into an azole-sensitive species are a potentially important new azole resistance mechanism. IMPORTANCE Azole antifungals are the main treatment for fungal disease in humans. Many species are intrinsically resistant to azoles-in other words all members of the species are resistant without prior exposure-and we do not understand why. In this study, we serendipitously discovered that many intrinsically resistant species have alternative or extra copies of the azole target gene, CYP51. Transfer of one of these genes from a resistant species to a sensitive one resulted in drug resistance, showing that the extra copies of CYP51 can confer drug resistance. Understanding how clinically important species are resistant to therapy allows us to predict whether a species could be resistant from genome sequence.

Antimicrobial and antioxidant activities of silver nanoparticles synthesized by novel biogenic method using mixed reductants.

A novel method, for the synthesis of silver nanoparticles that are eco-friendly by means of mixed reductants method, has been developed. The combined extract of Mentha viridis plant and Prunus domestica gum were used as reducing agents for the synthesis of silver nanoparticles of the size less than 40 nm in diameter. The effect of time and concentration on the formation of silver nanoparticles were also monitored. The silver nanoparticles formed were verified by surface Plasmon spectra using single and double beam UV-Vis spectrophotometer. The XRD technique and scanning electron microscopy were performed to analyze the crystalline structure, crystallite size and morphology. The synthesized silver nanoparticles were tested against different bacterial and fungus strains. The silver nanoparticles showed good inhibition in antimicrobial study and low MIC for bacterial strains. The antioxidant assay was performed to check the scavenging activity. In DPPH, the silver nanoparticles showed good scavenging activity and were found close to that of ascorbic acid.

Striking Back against Fungal Infections: The Utilization of Nanosystems for Antifungal Strategies.

Fungal infections have become a major health concern, given that invasive infections by Candida, Cryptococcus, and Aspergillus species have led to millions of mortalities. Conventional antifungal drugs including polyenes, echinocandins, azoles, allylamins, and antimetabolites have been used for decades, but their limitations include off-target toxicity, drug-resistance, poor water solubility, low bioavailability, and weak tissue penetration, which cannot be ignored. These drawbacks have led to the emergence of novel antifungal therapies. In this review, we discuss the nanosystems that are currently utilized for drug delivery and the application of antifungal therapies.

Synthesis, Identification, and Biological Study for Some Complexes of Azo Dye Having Theophylline.

This article includes the synthesis of heterocyclic azo dye of theophylline by coupling diazonium salt of 4-chloroaniline with theophylline which is, namely, 8-(1-(4-chlorophenyl)azo)theophylline (CPAT). The complexes of cobalt and nickel were prepared by reacting their ions with CPAT ligand in ethanol under 1 : 2 ratio metal-ligand. The CPAT ligand and its complexes were characterized by elemental analysis, infrared spectrometry, electronic absorption spectroscopy, molar conductivity, and magnetic moment. The cobalt and nickel complexes show octahedral geometry having general formula [M(CPAT)2Cl2]. This article addresses the properties of CPAT dye such as photochromic properties. The CPAT dye exhibited obvious and desired changes under irradiation with visible light (405 nm), high sensitive for pH changes which refer to its ability to be analysis indicator. CPAT dye exhibited solvatochromic properties presenting red shift with polar solvent. The CPAT and its complexes show interesting antibiological activities towards Staph. aureus and E. coli bacteria and Aspergillus fungi.

Molecular typing and antifungal susceptibility study of Aspergillus spp. in intensive care unit (ICU) patients in Indonesia.

INTRODUCTION: Aspergillus exhibits a wide variation of susceptibility against antifungals according to genetic and environmental factors. Identification to the species level is necessary for appropriate treatment. Our objective was to determine the Aspergillus species involved in invasive pulmonary aspergillosis (IPA) among ICU patients in Jakarta, Indonesia. METHODOLOGY: The incidence of IPA in ICU patients at six hospitals in Jakarta from October 2012 - January 2015 was investigated. It involved a collection of endotracheal aspirates (ETA), nasal swabs and environmental samples around the hospitals, phenotypic screening, molecular characterization, and antifungal susceptibility testing. RESULTS: Of the 405 patients investigated, 31 patients (7.7%) were diagnosed with putative IPA, from whom 45 Aspergillus isolates were collected. Aspergillus isolates were identified from pulmonary secretions in 24 patients, from nasal swabs in 7 patients and from both pulmonary secretions and nasal swabs in 7 patients. The phenotypic method showed 33 isolates of Aspergillus flavus (73.4%), nine Aspergillus fumigatus (20%), two Aspergillus niger (4.4%), and one Aspergillus nidulans (2.2%) isolate. Molecular identification showed 27 isolates of A. flavus (60.0%), eight isolates of A. fumigatus (17.8%), two isolates of A. niger (4.4%) and one isolate of A. nidulans (2.2%), while seven isolates (15.6%) were cryptic species or mixed isolates. CONCLUSIONS: Susceptibility testing showed all isolates were susceptible to amphotericin B, azoles and micafungin. Aspergillus flavus was the main causative organism in IPA cases in Jakarta, followed by A. fumigatus.