The current state of Clinical Mycology in Eastern and South-Eastern Europe.

The ability of medical centers in Eastern and South-Eastern Europe to diagnose and treat fungal infections remains unknown. In order to investigate that, here we conducted a cross-sectional online survey, released at both The International Society for Human & Animal Mycology (ISHAM) and European Confederation of Medical Mycology (ECMM) websites. A total of 31 institutions responded to the questionnaire. Most centers (87.1%, n = 27) had access to Aspergillus spp. ELISA galactomannan testing as well as to Cryptococcus spp. antigen testing (83.9%, n = 26). Serological tests were mostly available for Aspergillus species (80.6%, n = 25); and most institutions reported access to mold-active antifungal drugs (83.9%; n = 26), but 5-flucytosine was available to only 29% (n = 9) of the participant centers. In conclusion, this study represents the first attempt to document the strengths and limitations of the Eastern and South-Eastern European region for diagnosing and treating fungal diseases. LAY SUMMARY: Our article is about the availability of diagnostic and treatments tools related to fungal infections in the countries of Eastern and South-Eastern region. Surveys like these are important to understand the gaps and point towards the fungal infections as a global health issue.

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.

Chronic rhinosinusitis: association of recalcitrant nasal polyposis and fungal finding in polyp's single-cell suspension.

In recent years fungi are favoured as origin of chronic rhinosinusitis (CRS), especially with nasal polyps (wNP). Sensitive methods for fungal detection are still absent, therefore we used NP tissue single-cell suspension for mycology investigations in patients with recalcitrant NP (rNP) that underwent functional endoscopic sinus surgery (FESS). A prospective case-series study and culture-based mycological examination were conducted in patients who underwent FESS for the first time (ft-FESS) and those with repeated FESS (re-FESS). The study was conducted in a tertiary Otorhinolaryngology Unit of Clinical Centre of Serbia. A total of 43 consecutive patients with CRSwNP underwent FESS. Culture-based mycological examination of single-cell suspension was done on 55 NPs samples. Patient's co-morbidity data were collected. Repeated FESS was observed in 19/43 (44 %) patients (re-FESS group). Asthma and aspirin intolerance were more frequent in re-FESS than in ft-FESS group (p = 0.000, p = 0.002; respectively). Fungi were detected (wF) in 10/43 (23.3 %) patients (FESSwF group), representing 13/55 culture positive NP tissue (23.6 %). Fungal presence was higher in re-FESS than in ft-FESS group (42 and 8 %, respectively; p = 0.01). Significantly longer duration of CRS was observed in FESSwF than in fungal negative patients (p = 0.033). Predominate strain was Aspergillus flavus detected in 6/10 patients. This is the first study which analysed association of fungi in single-cell suspension of NP tissue and rNP. We demonstrate significantly higher percentage of positive fungal finding in re-FESSwF than in ft-FESSwF group. The most commonly isolated species in our patients was A. flavus.

European candidaemia is characterised by notable differential epidemiology and susceptibility pattern: Results from the ECMM Candida III study.

The objectives of this study were to assess Candida spp. distribution and antifungal resistance of candidaemia across Europe. Isolates were collected as part of the third ECMM Candida European multicentre observational study, conducted from 01 to 07-07-2018 to 31-03-2022. Each centre (maximum number/country determined by population size) included ∼10 consecutive cases. Isolates were referred to central laboratories and identified by morphology and MALDI-TOF, supplemented by ITS-sequencing when needed. EUCAST MICs were determined for five antifungals. fks sequencing was performed for echinocandin resistant isolates. The 399 isolates from 41 centres in 17 countries included C. albicans (47.1%), C. glabrata (22.3%), C. parapsilosis (15.0%), C. tropicalis (6.3%), C. dubliniensis and C. krusei (2.3% each) and other species (4.8%). Austria had the highest C. albicans proportion (77%), Czech Republic, France and UK the highest C. glabrata proportions (25-33%) while Italy and Turkey had the highest C. parapsilosis proportions (24-26%). All isolates were amphotericin B susceptible. Fluconazole resistance was found in 4% C. tropicalis, 12% C. glabrata (from six countries across Europe), 17% C. parapsilosis (from Greece, Italy, and Turkey) and 20% other Candida spp. Four isolates were anidulafungin and micafungin resistant/non-wild-type and five resistant to micafungin only. Three/3 and 2/5 of these were sequenced and harboured fks-alterations including a novel L657W in C. parapsilosis. The epidemiology varied among centres and countries. Acquired echinocandin resistance was rare but included differential susceptibility to anidulafungin and micafungin, and resistant C. parapsilosis. Fluconazole and voriconazole cross-resistance was common in C. glabrata and C. parapsilosis but with different geographical prevalence.

Anticryptococcal cytotoxicity of murine nonadherent cells is perforin and nonperforin mediated.

The encapsulated fungal pathogen Cryptococcus neoformans is a significant agent of life-threatening infections, particularly in people with suppressed cell-mediated immunity. The cellular cytotoxicity against C. neoformans infection is mainly mediated by NK and T cells, but effector mechanisms are not well understood. The objective of this study was (i) to determine whether prior exposure to the cryptococcal antigens enhances anticryptococcal activity of cytotoxic cells in mice and (ii) the contribution of perforin- and nonperforin-mediated cytotoxicity of NK and T cells in growth inhibition of C. neoformans. Our data showed that in vitro exposure of nonadherent (NA) spleen mononuclear cells from nonimmunized mice to heat-killed C. neoformans strain Cap67 unencapsulated mutant of B3501 (Ag1) or its supernatant (Ag2) demonstrated higher anticryptococcal activity. This effector mechanism can be enhanced further after immunization with either Ag1 or Ag2. There is a synergistic effect of immunization and in vitro incubation of the NA cells with the same antigens. Concanamycin A (CMA) and strontium chloride (SrCl2) inhibition assays were performed to clarify the contribution of perforin- and nonperforin-mediated anticryptococcal cytotoxicity of NA cells in these events. Treatment with these inhibitors demonstrated that anticryptococcal cytotoxicity of nonprimed NA cells was primarily perforin mediated. Anticryptococcal activity of the NA cells obtained from immunized mice after in vitro incubation with cryptococcal antigens was both perforin and nonperforin mediated. Taken together these data demonstrate that in mice a nonperforin-mediated pathway of anticryptococcal cytotoxicity can be induced by immunization. Further research is needed to examine their potential role for human vaccines strategies and/or therapies.

Aspergillus fumigatus branching complexity in vitro: 2D images and dynamic modeling.

BACKGROUND: Aspergillus fumigatus causes serious infections in humans, and its virulence correlates with hyphal growth, branching and formation of the filamentous mycelium. The filamentous mycelium is a complex structure inconvenient for quantity analysis. In this study, we monitored the branching of A. fumigatus filamentous mycelium in vitro at different points in time in order to assess the complexity degree and develop a dynamic model for the branching complexity. METHOD: We used fractal analysis of microscopic images (FAMI) to measure the fractal dimensions (D) of the branching complexity within 24 h of incubation. RESULTS: By photographing the filamentous mycelium dynamically and processing the images, the D variation curve of A. fumigatus complexity degree was obtained. We acquired the D variation curve which contained initial exponential period and stationary period of A. fumigatus branching. Further, the obtained data of D was modeled via the logistic model (LM) to develop a dynamic model of A. fumigatus branching for the prediction of the specific growth rate of branching value (0.23 h-1). CONCLUSIONS: Developed FAMI and LM models present a simple and non-destructive method of predicting the evolution of branching complexity of A. fumigatus. These models are useful as laboratory measurements for the prediction of hyphal and mycelium development, especially relevant to the pathogenesis study of aspergillosis, as well as pathogenesis of other diseases caused by moulds.

Estimated Burden of Serious Fungal Diseases in Serbia.

For the first time, we aimed to estimate the burden of serious fungal infections or diseases (SFD) and highlight national epidemiological features in Serbia. Data on population and underlining conditions were extracted from the Statistical Office of the Republic of Serbia, World Bank, the Institute of Public Health of Serbia, the World Health Organization, National reference laboratory for medical mycology, the national registries of Serbian professional societies, and relevant publications. The population structure/inhabitants in 2016 (not including the autonomous region Kosovo & Metohija) was 7,058,322; with 6,041,743 adults (85.6%). The populations at risk (total cases per year) were: HIV infected 2441; acute myeloid leukemia 212; stem cell transplantation 151; solid organ transplants 59; chronic obstructive pulmonary disease 250,302; adult asthmatics 311,806; adult cystic fibrosis 65; pulmonary tuberculosis 898; lung cancer 7260; intensive care unit admissions 19,821; and renal support 520. Annual fungal disease cases estimated are: candidemia 518; invasive aspergillosis 619; Candida peritonitis 187; Pneumocystis jirovecii pneumonia 62; cryptococcosis 5; mucormycosis or fusariosis 23; severe asthma with fungal sensitization 10,393; allergic bronchopulmonary aspergillosis 9094; chronic pulmonary aspergillosis 448, recurrent Candida vaginitis 135,303; oral candidiasis 208,489; esophageal candidiasis 173, fungal keratitis 70; tinea capitis 300; and onychomycosis 342,721. We expect that 156,825 people suffer from serious SFD each year (2221/100,000), and 409 dies annually. Additionally, the prevalence of superficial infections exceeds 1,008,995 cases (14,295/100,000). The first Rhinosporidium outbreak in Europe was associated with Serbian Silver Lake. The plant pathogen Fusarium seems to be emerging in Serbian pediatric haematooncology settings. Candida auris and endemic mycoses have not been observed to date. These general estimates provide a primer for further efforts to study fungal epidemiology in Serbia.

[Favorable Outcome of Hepatosplenic Candidiasis in a Patient with Acute Leukemia].

INTRODUCTION: Acute leukemias treatment requires strong chemotherapy. Patients that develop bone marrow aplasia become immunocompromised, thus becoming liable to bacterial and fungal infections. Fungal infections caused by Candida are frequent. Hepatosplenic candidiasis (HSC) is a frequent consequence of invasive candidiasis which is clinically presented with prolonged febrility unresponsive to antibiotics. CASE OUTLINE: A 53-year-old patient with acute myeloid leukemia was submitted to standard chemotherapy "3+7" regimen (daunoblastine 80 mg i.v. on days 1 to 3, cytarabine 2 x 170 mg i.v. during 7 days) and achieved complete remission. However, during remission he developed febrility unresponsive to antibiotics. Computerised tomography (CT) of the abdomen showed multiple hypodense lesions within the liver and spleen. Haemocultures on fungi were negative. However, seroconversion of biomarkers for invasive fungal infection (FI) (Candida and Aspergillus antigen/Ag and antibody/Ab) indicated possible HSC. Only high positivity of anti-Candida IgG antibodies, positivity of mannan and CT finding we regarded sufficient for the diagnosis and antimycotic therapy.Three months of treatment with different antimycotics were necessary for complete disappearance of both clinical symptoms and CT findings. CONCLUSION: In patients with prolonged febrile neutropenia IFI has to be strongly suspected. If imaging techniques show multiple hypodense lesions within liver and spleen, HSC has to be taken seriously into consideration. We believe that, along with CT finding, positive laboratory Candida biomarkers (mannan and IgG antibodies) should be considered sufficient for"probable HSC" and commencement of antifungal therapy, which must be long enough, i.e. until complete disappearance of clinical symptoms and CT findings are achieved.

Chronic necrotizing pulmonary aspergillosis in an immunocompetent, obese 10-year-old boy.

Chronic necrotizing pulmonary aspergillosis is a rare form of pulmonary aspergillosis. It is usually seen in middle-aged or elderly patients with underlying chronic lung disease or mild immunodeficiency, and has been only rarely encountered in children. Clinical presentation is variable and usually involves constitutional symptoms of several months' duration as well as respiratory symptoms. We present a previously well, immunocompetent, obese 10-year-old boy with cough and mild hemoptysis lasting for a couple of days and a round pulmonary infiltrate on chest radiograph. Further diagnostic investigations revealed the histopathological features of chronic necrotizing pulmonary aspergillosis in excised lung tissue, and Aspergillus fumigatus was isolated in lung tissue culture. This is one of the youngest described patients with this semi-invasive form of aspergillosis.

[Allergic fungal sinusitis--new aspects of clinical features, laboratory diagnosis and therapy].

Allergic fungal sinusitis (AFS) is a chronic non-invasive disease. Hypersensitive immune response is usually initiated by allergens of filamentous fungi Aspergillus, Penicillium, Cladosporium, Fusarium, Bipolaris, Curvularia and Alternaria. AFS is a clinical and immune analogue of the allergic bronchopulmonary aspergillosis (ABPA) as the sinus exudate resembles that of the bronchoalveolar lavage (BAL) in ABPA. Patients with AFS are usually immunocompetent, atopic and males. The most common symptoms are headache, fullness in the paranasal sinuses, and difficult breathing through the nose. Clinically, there is a chronic mucosal inflammation and histopathologic finding shows allergic mucin and eosinophils. Specific staining methods, Gomori's Methenamine Silver (GMS) or periodic acid-Schiff (PAS), are used for microscopic visualisation of hyphae, which are, in addition to the isolated fungi, most reliable evidence of AFS. Computerized tomography (CT) of paranasal sinuses shows the areas of hyperdensity. In cases where AFS is complicated by the erosion of bone tissue, discontinuation of the sinus bone wall can be seen. Significant laboratory finding, which correlate highly with the AFS, are high immunoglobulin E (IgE) antibodies specific for fungi, detected by the skin prick test or in serum. Treatment is often surgical, and after removal of the allergic mucin, therapy involves oral and nasal corticosteroids, immunotherapy and locally applied antimycotics (with verified fungal etiology). During treatment, the total/specific IgE is monitored--concentration increases with the development of AFS, and decreases during the improvement process. Knowledge of the pathophysiological mechanisms of AFS is scarce, and represents the focus of further research in order to define an optimal diagnostic and therapeutic approach.