Marina Pekmezovic

Molecular epidemiology and antifungal susceptibility of Serbian Cryptococcus neoformans isolates

Molecular typing and antifungal susceptibility testing of 34 clinical Serbian Cryptococcus neoformans isolates from 25 patients was retrospectively performed. Amplified fragment length polymorphism (AFLP) fingerprinting was used for genotyping, whereas a novel real‐time PCR was used to determine the mating‐ and serotype. The antifungals amphotericin B, 5‐fluorocytosine, fluconazole, voriconazole, itraconazole and posaconazole were used to determine the antifungal susceptibility profiles. The majority of isolates belonged to genotype AFLP1/VNI (n = 20; 58.8%), followed by AFLP2/VNIV (n = 10; 29.4%), AFLP3/VNIII (n = 3; 8.8%) and AFLP1B/VNII (n = 1; 2.9%). All AFLP1/VNI isolates were mating–serotype αA, the sole AFLP1B/VNII isolate was found to be aA, whereas AFLP2/VNIV harboured serotype D isolates with either the a (n = 2; 5.9%) or α (n = 8; 23.5%) mating‐type allele. The isolates (n = 3; 8.8%) that were found to be genotype AFLP3/VNIII had the hybrid mating‐ and serotype combination aA‐αD. In vitro antifungal susceptibility testing showed that all isolates were susceptible to amphotericin B, voriconazole and posaconazole. Low resistance level was observed for fluconazole (n = 1; 2.9%) and 5‐fluorocytosine. (n = 2; 5.8%). A large percentage of isolates was found to be susceptible dose dependent to itraconazole (n = 16; 47.1%). AFLP1/VNI was the most common genotype among clinical C. neoformans isolates from immunocompromised patients in Serbia. C. neoformans from HIV‐negative patients were significantly less susceptible to 5‐fluorocytosine (P < 0.01). Correlation between genotypes and antifungal susceptibility was not observed.

Importance of Resolving Fungal Nomenclature: the Case of Multiple Pathogenic Species in the Cryptococcus Genus

Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. ABSTRACT Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16 ), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature “C. neoformans species complex” and “C. gattii species complex.” Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

In vitro antifungal activities of amphotericin B, 5-fluorocytosine, fluconazole and itraconazole against Cryptococcus neoformans isolated from cerebrospinal fluid and blood from patients in Serbia.

Recently, geographic variations in resistance to agents commonly used in the treatment of cryptococcosis have been reported. Therefore, the antifungal susceptibilities of 31 clinical isolates of Cryptococcus neoformans, collected in Serbia during 10-year period, were investigated. Strains were isolated from cerebrospinal fluid (n=28) and blood (n=3), from patients with AIDS (n=26), lymphoma (n=4) and kidney transplant recipient (n=1). The minimal inhibitory concentrations (MICs) of amphotericin B, 5-fluorocytosine, fluconazole and itraconazole were determined by the E-test(®) method. The isolates were highly susceptible to amphotericin B (100% susceptibility at MIC<0.5 μg/mL) and 5-fluorocytosine (87.1% susceptibility at MIC ≤ 4 μg/mL). Geometric mean MIC of amphotericin B and 5-fluorocytosine were 0.102 μg/mL and 0.396 μg/mL, respectively. Fluconazole exhibited the lowest activity in vitro (48.4% susceptibility at MIC ≤ 8 μg/mL) with a significant resistance rate. The activity of itraconazole was also decreased (48.4% susceptibility at MIC ≤ 0.25 μg/mL). The geometric mean MIC of fluconazole stood at 15.14 μg/mL and of itraconazole was 0.144 μg/mL. Cross-resistance among azoles was not common (3.2%), but the parallel increase in fluconazole and itraconazole MIC has been observed (P<0.01). The low rate of susceptibility to fluconazole stresses the need for active antifungal surveillance of C. neoformans and of the corresponding data from different geographic regions.

The prevalence of Candida onychomycosis in Southeastern Serbia from 2011 to 2015.

Despite the increasing of onychomycosis caused by Candida spp., in referent literature, there is still data insufficiency about this nail infection. The objectives of this retrospective study were to determine epidemiological characteristics of Candida onychomycosis, the antifungal susceptibility of isolated species in vitro, and to compare the results of antifungal susceptibility testing with conducted treatment in period from 2011 to the end of March 2015. Out of 761 patients who were underwent clinical and mycological examinations, 137 had Candida species isolated from nails. The dominant species was Candida albicans (C. albicans) (36.59%) followed by C. parapsilosis (23.78%), C. krusei (9.76%), and C. guilliermondii (6.71%). Antifungal susceptibility in vitro testing showed good susceptibility to antimycotics, except C. krusei, which was resistance to fluconazole (FCZ) and isolates of C. tropicalis and C. glabrata which were dose dependent to itraconazole (ITZ) and fluconazole. Evaluation of medical histories determined that combined therapy, which included pulsed systemic regimen of ITZ with topical application of clotrimazole, had better clinical outcomes regarding the proscribed only topical application of clotrimazole. Multidisciplinary approach of dermatologists and mycologists is required in solving the problem of onychomycosis, which is the dominant nail disease.

Presence, species distribution, and density of Malassezia yeast in patients with seborrhoeic dermatitis – a community‐based case–control study and review of literature

Malassezia yeast belongs to the normal cutaneous flora and under certain conditions it causes seborrhoeic dermatitis (SD). There is no culture‐based study about the presence and density of the Malassezia in SD patients in Serbia. Aim was to show the presence, species distribution and density of Malassezia in patients with SD on lesional skin (LS) and non‐lesional skin (NLS) and healthy controls (HC) and to compare data between Serbia and other countries. The study included 70 HC and 60 patients with SD in the study group (SG). Isolation, identification and examination of density of Malassezia colony‐forming units from LS and NLS were performed. Malassezia was found more frequently in the SG than in HC, 90% and 60%, respectively (P < 0.01). The most frequent isolates in SG on LS were M. slooffiae (26%), followed by M. globosa (17%) and M. sympodialis (17%). The yeast density was much higher on LS of SG than on NLS of SG or in the HC group (P < 0.05). Higher density of Malassezia was shown on LS of SG than on NLS of SG and HC. M. slooffiae is the most prevalent species in SD patients in Serbia. This study demonstrated a positive relationship between severity of SD and presence of Malassezia spp.

Synthesis and evaluation of thiophene-based guanylhydrazones (iminoguanidines) efficient against panel of voriconazole-resistant fungal isolates.

A series of new thiophene-based guanylhydrazones (iminoguanidines) were synthesized in high yields using a straightforward two-step procedure. The antifungal activity of compounds was evaluated against a wide range of medicaly important fungal strains including yeasts, molds, and dermatophytes in comparison to clinically used drug voriconazole. Cytotoxic properties of compounds were also determined using human lung fibroblast cell line and hemolysis assay. All guanylhydrazones showed significant activity against broad spectrum of clinically important species of Candida spp., Aspergillus fumigatus, Fusarium oxysporum, Microsporum canis and Trichophyton mentagrophytes, which was in some cases comparable or better than activity of voriconazole. More importantly, compounds 10, 11, 13, 14, 18 and 21 exhibited excellent activity against voriconazole-resistant Candida albicans CA5 with very low minimal inhibitory concentration (MIC) values <2 μg mL(-1). Derivative 14, bearing bromine on the phenyl ring, was the most effective compound with MICs ranging from 0.25 to 6.25 μg mL(-1). However, bis-guanylhydrazone 18 showed better selectivity in terms of therapeutic index values. In vivo embryotoxicity on zebrafish (Danio rerio) showed improved toxicity profile of 11, 14 and 18 in comparison to that of voriconazole. Most guanylhydrazones also inhibited C. albicans yeast to hyphal transition, essential for its biofilm formation, while 11 and 18 were able to disperse preformed Candida biofilms. All guanylhydrazones showed the equal potential to interact with genomic DNA of C. albicans in vitro, thus indicating a possible mechanism of their action, as well as possible mechanism of observed cytotoxic effects. Tested compounds did not have significant hemolytic effect and caused low liposome leakage, which excluded the cell membrane as a primary target. On the basis of computational docking experiments using both human and cytochrome P450 from Candida it was concluded that the most active guanylhydrazones had minimal structural prerequisites to interact with the cytochrome P450 14α-demethylase (CYP51). Promising guanylhydrazone derivatives also showed satisfactory pharmacokinetic profile based on molecular calculations.

Functionalised isocoumarins as antifungal compounds: Synthesis and biological studies.

A series of novel 3-substituted isocoumarins was prepared via Pd-catalysed coupling processes and screened in vitro for antifungal activity against Candida species. The study revealed antifungal potential of isocoumarins possessing the azole substituents, which, in some cases, showed biological properties equal to those of clinically used voriconazole. Selected compounds were also screened against voriconazole resistant Candida krusei 6258 and a clinical isolate Candida parapsilosis CA-27. Although the activity against these targets needs to be improved further, the results emphasise additional potential of this new class of antifungal compounds.

In vitro Protease Inhibition and Cytotoxicity of Aspergillus fumigatus Biomolecules Secreted under Long-Term Aerated Conditions

The fatality rate of invasive aspergillosis (IA) is still very high, especially in prolonged and untreated pulmonary cases. Aspergillus fumigatus is the main causative agent of IA and investigation of its metabolites could provide valuable insight into virulence factor(s) associated with this organism. We evaluated the A. fumigatus culture filtrate (CF) products generated during short- and long-term aerated and non-aerated conditions and tested for (i) inhibition of cysteine or serine proteases and (ii) cytotoxicity. In addition, the mathematical model was determined using response surface methodology (RSM) to estimate the influence of different fermentation conditions on A. fumigatus CF characteristics, predict enzyme inhibition and make possible correlations with in vivo conditions. Biosynthesis of A. fumigatus low molecular weight proteinaceous products (from 6.4 to 15.4 kDa) was observed after 6 days of growth under aerated and alkaline conditions. Also, only these CFs showed significant reduction in cell lines survival (Caco-2 and WISH 35.6% and 54.6%, respectively). Obtained results provide solid starting point for further studies that would include: (i) detailed chemical characterization of A. fumigatus CF, (ii) activity relationships and in vivo correlation with pathogenicity of prolonged pulmonary IA and (iii) possible use of biomolecules as diagnostic or therapeutic markers.

Control of human and plant fungal pathogens using pentaene macrolide 32, 33-didehydroroflamycoin

The aim of this study was to address the toxicity of recently described polyene macrolide 32, 33‐didehydroroflamycoin (DDHR) on a wide range of fungal pathogens and its potential to control plant fungal diseases.

Susceptibility profile of Candida spp. isolated from humans and dogs with stomatitis to the essential oil of Thymus vulgaris

Candida spp. form a part of human and animal oral cavity flora. However Candida spp. is the main cause of dental related stomatitis in humans and stomatitis in dogs. Stomatitis treatment implies the use of azoles and polyenes to which yeasts build up resistance. The research is directed to the use of natural compounds such as essential oils. The aim of this paper is to define the antifungal activity of thyme oil on 15 clinical strains of Candida spp., isolated from humans and dogs and to determine if there is a difference in susceptibility between human and dog isolates. Sampling in patients with stomatitis was done by swabbing the denture or oral mucosa swab while sampling in dogs was done by swabbing the oral cavity mucosa after stomatitis has been diagnosed. In order to investigate the antifungal activity of thyme oil in vitro, microdilution method was used. Thyme oil expressed antifungal effects on all investigated strains. Also, our data show that the values of minimum fungicide concentration (MFC) and minimum inhibitory concentration (MIC) are lower in human strains. Explanation is that in most cases, stomatitis in humans is asymptomatic and thus not treated, so Candida strains have not developed resistance. On the other hand, stomatitis in dogs is followed by a marked clinical picture and treated is by antimicotics (mostly by azoles), therefore resistant Candida strains are more likely to occur.