Valentina S Arsic Arsenijevic

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.

A laboratory-based study on patients with Parkinson’s disease and seborrheic dermatitis: the presence and density of Malassezia yeasts, their different species and enzymes production

BackgroundSeborrheic dermatitis (SD) and Parkinson’s disease (PD) are frequently associated conditions. Aims of this study were: to determine severity of SD, presence of different species and density of Malassezia yeasts; to assess yeast lipases and phosphatases production in vitro and to compare these results between SD patients with and without PD.MethodsThis case–control prospective study was conducted at the Dermatology and Neurology Units, Clinical Centre of Serbia and at the National Medical Mycology Reference Laboratory, University of Belgrade Medical School, Serbia. A total of 90 patients and 70 healthy controls (HC) were investigated: 60 patients with SD (SDN) and 30 patients with SD and PD (SDP). Culture-based mycological examination was carried out on lesional skin (LS) and non-lesional skin (NLS). A yeasts density was determined by counting the Malassezia colony forming units per tape (CFU/tape). Enzymes production by isolated Malassezia was investigated.ResultsThe most patients with SD were male (76.7%; SDP and 63.3%; SDN) and the intensity of SD was dominantly severe or moderate (76.7%; SDP and 75%; SDN). The presence of Malasseziа was high on LS in both groups (87.3%; SDP and 86.7%; SDN) (p=0.667).The highest yeasts density (mean CFU/tape=67.8) was detected on LS in 53% of SDP group and in 21.7% of SDN group (mean CFU/tape=31.9) (p < 0.01). The presence of negative cultures was lower in SDP group (13.3%) in comparison to HC and SDN groups (37% and 31.7%, respectively). Malassezia density on NLS in SDP group (mean CFU/tape=44.3) was significantly higher in comparison to SDN and HC (p=0.018). M. globosa was the most abundant species identified amongst isolates from the SDP group (42.3%) and exhibited high production of phosphatase and lipase in vitro.ConclusionFrom this laboratory-based study a positive correlation between SD, PD, M. globosa incidence, high yeast density and high phosphatase and lipase activity was established. Our data lead to conclusion that local skin performance of PD patient’s characterized with increased sebum excretion ratio play a role in SD by stimulation of yeasts replication and enzyme production.

What lurks in the sellar

In June, 2008, a 44-year-old immunocompetent man presented to us with sinusitis associated with headache, transient diplopia, and dizziness. He had a 7-year history of chronic sinusitis occasionally treated with pseudoephedrine and antibiotics. CT of the sinuses showed opacifi cation of the paranasal sinuses which were fi lled with high density material, erosion of the sphenoid bone, and propagation of the pathological process to the sellar region, with a large sellar mass (fi gure A). MRI confi rmed a giant hypointense lesion in the sellar region. The pituitary gland was displaced upwards. Physical examination was unremarkable, except for obesity (BMI 38 kg/m2). Computerised visual-fi eld examination, and visual acuity and motility were normal. The insulin hypoglycaemia test showed a peak growth hormone (GH) of 3·4 μg/L, borderline for severe GH defi ciency (<3·0 μg/L); cortisol response was normal. Prolactin concentration was high (1150 mU/L; mean of three values). Macroprolactin was negative. Testosterone, FT4, and TSH concentrations were normal, and antibodies to thyroid peroxidase and thyroglobulin were present, compatible with a diagnosis of euthyroid chronic thyroiditis. In view of the large cystic sellar mass, and to provide symptomatic relief, the patient underwent transphenoidal surgery. Intraoperatively, thick brown material with a peanut-butter-like consistency was cleared from the sella. Histological examination showed many eosinophils within the eosinophilic mucin, but did not show a pituitary adenoma (fi gure B). In addition, septate fungal hyphae with acute angle branching embedded within the clusters of eosinophils were seen, with no fungal invasion of the tissue (fi gure C). Culture of the sellar material grew a white cotton-like mould colony; microscopically, hyphae with spicules and clamp connections were seen, suggestive of Schizophyllum commune. This was confi rmed by sequence analysis (strain number CBS 124811). Despite pituitary stalk decompression, prolactin concentrations remained high (990 mU/L). For the residual mass in the sinuses, functional endoscopic sinus surgery was done. The patient was treated with systemic antimycotics (liposomal amphotericin B, 2 weeks before and 1 week after surgery) and systemic and topical corticosteroids, followed by itraconazole for 2 months). When seen in August, 2009, he had improved clinically and prolactin concentration had decreased to 550 mU/L. We regularly inhale spores of diverse fungal species, yet fungal disease is uncommon. Fungal organisms within nasal mucin are present in up to 93% of patients with chronic sinusitis and even in healthy persons without sinonasal disease. Allergic fungal sinusitis is a noninfectious chronic disease with an immunological response to colonising fungi resulting in an eosinophilic infl ammation. S commune is a basidiomycetes fungus which has been identifi ed in some verifi ed human infections. In our case, S commune triggered an aggressive eosinophilic infl ammation. The diff erential diagnosis of large sellar masses relies mainly on pathological analysis. The most common lesions encountered in the sellar region are infective, vascular, neoplastic, or immunological. The giant hypointense lesion in the sella on MRI scans results from the very high amounts of proteins released by the eosinophils into the mucin. CT is a better diagnostic modality for allergic fungal sinusitis. In our patient, the persistence of hyperprolactinaemia after pituitary surgery, with no compression to the pituitary stalk, was intriguing. In-vitro studies show that fungal glucans may directly interact with glucan-specifi c receptors on rodent somatomammotroph cell membranes to stimulate prolactin secretion. In view of the many individuals with chronic sinusitis, physicians should be vigilant in recognising and referring patients with aggressive disease to appropriate specialists.

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.

Epidemiology of onychomycosis in Serbia: a laboratory-based survey and risk factor identification

Onychomycosis is a fungal infection of the fingernails and/or toenails caused by dermatophytes, yeasts and non‐dermatophyte moulds. The epidemiology of onychomycosis in Serbia is yet to be fully established. This epidemiological study was aimed at evaluating the epidemiology of onychomycosis in a sample of the Serbian patients at risk of onychomycosis, to determine the fungal aetiological agents and to identify the possible risk factors. The study population included 374 patients from six centres in Serbia with suspected onychomycosis. Demographic data, data about comorbidities, lifestyle, clinical aspects of onychomycosis, trauma, excessive perspiration and personal and family history of previous onychomycosis were studied. Laboratory confirmation of diagnosis was done by direct microscopy, fungal culture and PCR. Diagnosis of onychomycosis was confirmed in 50.8% of patients, who tested positive to at least one laboratory test (direct microscopy, fungal culture or PCR). Trichophyton rubrum was predominant both on toenails (85.98%) and on fingernails (38.46%). Independent risk factors for onychomycosis were: old age (OR = 2.285; P < 0.001), family history of previous onychomycosis and/or tinea pedis (OR = 2.452; P = 0.005), excessive perspiration (OR = 2.165; P = 0.002) and higher degree of hyperkeratosis (OR = 1.755; P = 0.020). This is a first epidemiological study of onychomycosis from Serbia.

Mathematical modeling of the neuron morphology using two dimensional images.

In this study mathematical analyses such as the analysis of area and length, fractal analysis and modified Sholl analysis were applied on two dimensional (2D) images of neurons from adult human dentate nucleus (DN). Using mathematical analyses main morphological properties were obtained including the size of neuron and soma, the length of all dendrites, the density of dendritic arborization, the position of the maximum density and the irregularity of dendrites. Response surface methodology (RSM) was used for modeling the size of neurons and the length of all dendrites. However, the RSM model based on the second-order polynomial equation was only possible to apply to correlate changes in the size of the neuron with other properties of its morphology. Modeling data provided evidence that the size of DN neurons statistically depended on the size of the soma, the density of dendritic arborization and the irregularity of dendrites. The low value of mean relative percent deviation (MRPD) between the experimental data and the predicted neuron size obtained by RSM model showed that model was suitable for modeling the size of DN neurons. Therefore, RSM can be generally used for modeling neuron size from 2D images.

Surveillance and characterization of Candida bloodstream infections in a Serbian tertiary care hospital

INTRODUCTION Candida spp. frequently cause hospital-acquired bloodstream infections (BSI) with a high mortality rate (up to 70%). We analyzed the frequency, infection characteristics, potential predisposing factors, susceptibility to antifungal drugs, biofilm production and other virulence characteristics of Candida spp. isolates obtained from a tertiary care hospital in Niš, Serbia, during a one year period. METHODS Medical histories, characteristics of isolated strains and drug susceptibility, as well as the effect on the function of isolated macrophages and other virulence features were evaluated. The obtained results were subjected to students t-test and multivariate statistical analyzes. RESULTS Herein we report an annual incidence of 3.65 cases of C. albicans, C. lusitaniae and C. lipolytica infections per 105 population. Out of eight isolated strains, two (25%) were shown to be strong biofilm producers, one (12.5%) caused hemolysis on blood agar and in two (25%) cases macrophages were able to completely eliminate the yeast colonies. Chronic kidney disease, diabetes, malignant and other diseases were present in 37.5, 62.5, 50 and 75%, respectively, in the study group. All patients with Candida BSI received antifungal therapy (amphotericin B), however, hospital mortality was observed in 25% of patients. CONCLUSIONS Evaluation of local Candida epidemiology, antifungal susceptibility and virulence factors, as well as personalized patient risk factors are important for the surveillance of Candida BSI, especially in intensive care unit patients and may contribute to the improved options and outcome for patients with Candida BSI.

Candida bloodstream infections in Serbia: First multicentre report of a national prospective observational survey in intensive care units

Candida bloodstream infections (BSI) are a significant cause of mortality in intensive care units (ICU), hereof the prospective 12‐months (2014‐2015) hospital‐ and laboratory‐based survey was performed at the Serbian National Reference Medical Mycology Laboratory (NRMML). Candida identification was done by a matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry and a susceptibility test, according to the Clinical and Laboratory Standards Institute methodology. Among nine centres (265 beds; 10 820 patient admissions), four neonatal/paediatric (NICU/PICUs) and five adult centres (ICUs) participated, representing 89 beds and 3446 patient admissions, 166 beds and 7347 patient admissions respectively. The NRMML received 43 isolates, 17 from NICU/PICUs and 26 from adult ICUs. C. albicans dominated highly in NICU/PICUs (~71%), whereas C. albicans and C. parapsilosis were equally distributed within adults (46%, each), both accounting for ~90% of received isolates. The resistance to itraconazole and flucytosine were 25% and 2.4% respectively. In addition, the 2 C. albicans were azole cross‐resistant (4.6%). The overall incidence of CandidaBSI was ~3.97 cases/1000 patient admissions (4.93 in NICU/PICU and 3.53 in adult ICU). The 30‐day mortality was ~37%, most associated with C. tropicalis and C. glabrataBSI. Data from this national survey may contribute to improving the Balkan and Mediterranean region epidemiology of CandidaBSI within ICUs.

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.