Inhaler Steroid Use Changes Oral and Airway Bacterial and Fungal Microbiome Profile in Asthma Patients.

INTRODUCTION: The full spectrum of bacterial and fungal species in adult asthma and the effect of inhaled corticosteroid use is not well described. The aim was to collect mouthwash and induced sputum samples from newly diagnosed asthma patients in the pretreatment period and in chronic asthma patients while undergoing regular maintenance inhaled corticosteroid therapy, in order to demonstrate the bacterial and fungal microbiome profile. METHODS: The study included 28 asthmatic patients on inhaler steroid therapy, 25 steroid-naive asthmatics, and 24 healthy controls. Genomic DNA was isolated from induced sputum and mouthwash samples. Analyses were performed using bacterial primers selected from the 16S rRNA region for the bacterial genome and "panfungal" primers selected from the 5.8S rRNA region for the fungal genome. RESULTS: Dominant genera in mouthwash samples of steroid-naive asthmatics were Neisseria, Haemophilus, and Rothia. The oral microbiota of asthmatic patients on inhaler steroid treatment included Neisseria, Rothia, and Veillonella species. Abundant genera in induced sputum samples of steroid-naive asthma patients were Actinomyces, Granulicatella, Fusobacterium, Peptostreptococcus, and Atopobium. Sputum microbiota of asthma patients taking inhaler steroids were dominated by Prevotella and Porphyromonas. Mucor plumbeus and Malassezia restricta species were abundant in the airways of steroid-naive asthma patients. Choanephora infundibulifera and Malassezia restricta became dominant in asthma patients taking inhaled steroids. CONCLUSION: The oral and airway microbiota consist of different bacterial and fungal communities in healthy and asthmatic patients. Inhaler steroid use may influence the composition of the oral and airway microbiota.

[Investigation of Cryptococcus Colonization and Mating Genotype in Environmental Samples]. / Çevresel Örneklerde Cryptococcus Kolonizasyonunun ve Esey Genotipinin Arastirilmasi.

Cryptococcus species are fungal pathogens that pose a serious threat to human life and can cause meningoencephalitis in immunocompromised and healthy individuals. It was estimated that approximately 112000 people die every year due to cryptococcal-related infections all over the world, especially in immunocompromised individuals. Cryptococcus species can be found in soil, bat dung, pigeon droppings, and various tree species in addition to humans. Despite the majority of Cryptococcus species being haploid opportunistic human pathogens, it is known that the ability to undergo sexual reproduction plays a significant role in the expansion of species distribution and the increase in virulence. In Cryptococcus species, sexual reproduction is governed by the mating genotype gene region called the MAT locus. Pathogenic Cryptococcus species have two mating types (MATa and MATα), defined by the presence of one of two alternative alleles at a single MAT locus. In this study, various tree species (eucalyptus, olive and carob) in a total of seven regions in Mersin (Gülnar, Göksu, Narlikuyu, Ayas, Kizkalesi, and Tarsus) and Hatay provinces were examined to detect Cryptococcus species. The aim of this study was to determine the environmental distribution and sexual genotypes of Cryptococcus species in these regions. In the present study, samples were collected from a total of 750 trees, including olive, eucalyptus, and carob trees. The samples were incubated on Staib agar medium containing 0.1% biphenyl and 0.5% chloramphenicol. Colonies that formed brown pigment were identified as C.neoformans using conventional and molecular methods. The sexual genotypes were determined by comparing the lengths of the STE20 gene from the isolates compared with those of reference C.neoformans strains. Growth was observed in 97 (12.9%) of 750 samples collected from eucalyptus (n= 236), olive (n= 303) and carob (n= 211) trees. All 97 isolates were determined to be C.neoformans var. grubii. The highest positivity was found in Narlikuyu (78.2%), and from carob (9.4%) and olive (3.5%) trees. Cryptococcus species was not detected in any of the samples derived from eucalyptus trees. Based on the lengths of the STE20 gene, it was determined that all C.neoformans var. grubii isolates were in the MAT Aα genotype. The data obtained regarding the environmental distribution of Cryptococcus species and the distribution of genes involved in sexual reproduction are believed to provide valuable guidance in terms of the potential clinical implications of environmental Cryptococcus hotspots and regional species characteristics in our country.

[Evaluation of Malassezia furfur Biofilm Formation on Polypropylene Membrane]. / Polipropilen Membran Üzerinde Malassezia furfur Biyofilm Olusumunun Degerlendirilmesi.

The Malassezia yeast species colonize on the skin immediately after birth and could be found on the healthy skin flora for life. Although they are more frequently involved in the etiology of common skin infections in the community, particularly Malassezia furfur could cause life-threatening infections such as fungemia. Detection of biofilm during the colonization of these yeasts on the skin is an important criterion for its virulence. Since they are lipophilic yeasts, commonly used biofilm detection methods are not applicable to the Malassezia strains. The aim of the study was to describe the growth and measurement of M.furfur isolates on a polypropylene membrane to demonstrate their biofilm-forming capacities. Twenty-seven M.furfur strains colonized in the newborns were included in the study. Basically, sterile polypropylene membranes were placed on different polysorbates (tween 20, 40, and 80) which were spread on Sabouraud dextrose agar. Ten µl saline suspension of M.furfur was dropped on the polypropylene membrane and incubated in standard growth conditions for three days. Later, the visible colony was removed gently by washing with running water and the biofilm structure formed on the membrane was stained with safranin. The stained biofilm was photographed. Performing image analysis, the values obtained against background activity were digitized according to the specified protocol. Moreover, XTT reduction test was performed and the measured metabolic activity results were compared with the safranin-stained biofilm data. The safranin hydrolysis of the strains was measured spectrometrically. Twenty-five (92.6%) of the strains included in the study were stained with safranin, which indicated the presence of biofilm on the polypropylene membrane. The strains grown with tween 20 and tween 80 formed a higher biofilm layer density than those supplied with tween 40. Isolates with low and high biofilm-forming capacity were clearly separated by tween 20 (p< 0.05). XTT activity was detected in 26 (96.3%) isolates. No correlation was found between biofilm density obtained by the described method and XTT reduction. It was observed that hydrolysis of safranin did not affect the biofilm evaluation method. In this study, it was shown that as a result of sufficient diffusion through hydrophobic membranes, polysorbate-based growth factors could maintain measurement of the biofilm layer formed by lipophilic M.furfur strains. The best grouping properties for M.furfur were obtained with tween 20 which could determine low and high level of biofilm formation. Image analysis was used with high performance for this method. As conclusion, the utilization of different hydrophobic membranes and dyes would lead to the development of new techniques for the application in other lipophilic yeasts.

[Fungal Keratitis Associated with Curvularia lunata: First Case Report from Türkiye]. / Curvularia lunata'ya Bagli Gelisen Fungal Keratit: Türkiye'den Ilk Olgu Raporu.

Fungal keratitis is a medical emergency that is among the most common causes of blindness in developing countries. The type of the agent may vary depending on the geographical conditions under which the patient lives, trauma exposure, the use of contact lenses and profession. Curvularia spp. is a saprophytic genus that rarely causes systemic disease in humans and has 250 species identified to date. They proliferate in soil and plants and spread to the environment with their spores and the formation of blackish and fluffy colonies is its most well-known morphological feature. There may be difficulties in cultivating brown (dematiaceous) fungi. Due to the similarity between the genera, conventional methods remain inadequate for diagnosis. In this report, a case of fungal keratitis associated with C.lunata was presented. Seventy-five years-old female patient admitted to the hospital with the symptoms of stinging pain, blurred vision, and swelling in the right eye. Her symptoms had begun four days ago after her eye was hit by a plant. The patient who had a history of peripheral neuropathy due to diabetes mellitus (DM) was hospitalized with a preliminary diagnosis of keratitis, and in the cultures of the patient's corneal scraping samples, the filamentous, black pigment-forming colonies of the pathogen growing on 5% sheep blood agar and potato dextrose agar showing an aerial hyphal structure, were stained with lactophenol cotton blue and examined under the microscope. The microscopic examination revealed geniculate conidiophores with brown pigmentation. On top of these structures were tetralocular macroconidia, one of which appeared to be larger than the main axis. The fungus was subjected to molecular identification with the prediagnosis of Curvularia/Bipolaris. DNA extraction of the ITS region polymerase chain reaction amplification and Sanger sequencing were performed for molecular identification. Sanger sequencing identified the agent to be Curvularia lunata with a similarity rate of 99.79% (NCBI-GenBank Nucleotide ID: OR365075). In vitro antifungal susceptibility of C.lunata was evaluated by microdilution method. Itraconazole and amphotericin B showed higher activity against C.lunata compared to other antifungals while fluconazole was the least active antifungal. Intrastromal and subconjunctival voriconazole injection was applied to the patient who was unresponsive to empirically initiated oral moxifloxacin and different topical treatments (vancomycin, ceftazidime, flucanozole, ganciclovir, cyclopentolate hydrochloride, hyaluronic acid and trehalose). After injection, right penetrating keratoplasty was applied due to increased thinning of the ulcerated area. No pathogen was detected in cultures taken after keratoplasty. Rare fungi should be considered in cases of keratitis that are difficult to treat. Fungal keratitis caused by brown fungi are clinically similar to each other and effective treatment protocols cannot be implemented without a species identification. Identification of the pathogen will enable genus-specific treatment. This will also help prevent complications that may occur. This article aims to present a case of fungal keratitis associated with C.lunata.

Frequency of azole resistance in clinical and environmental strains of Aspergillus fumigatus in Turkey: a multicentre study.

OBJECTIVES: Aspergillus fumigatus causes several diseases in humans and azole resistance in A. fumigatus strains is an important issue. The aim of this multicentre epidemiological study was to investigate the prevalence of azole resistance in clinical and environmental A. fumigatus isolates in Turkey. METHODS: Twenty-one centres participated in this study from 1 May 2018 to 1 October 2019. One participant from each centre was asked to collect environmental and clinical A. fumigatus isolates. Azole resistance was screened for using EUCAST agar screening methodology (EUCAST E.DEF 10.1) and was confirmed by the EUCAST E.DEF 9.3 reference microdilution method. Isolates with a phenotypic resistance pattern were sequenced for the cyp51A gene and microsatellite genotyping was used to determine the genetic relationships between the resistant strains. RESULTS: In total, resistance was found in 1.3% of the strains that were isolated from environmental samples and 3.3% of the strains that were isolated from clinical samples. Mutations in the cyp51A gene were detected in 9 (47.4%) of the 19 azole-resistant isolates, all of which were found to be TR34/L98H mutations. Microsatellite genotyping clearly differentiated the strains with the TR34/L98H mutation in the cyp51A gene from the strains with no mutation in this gene. CONCLUSIONS: The rate of observed azole resistance of A. fumigatus isolates was low in this study, but the fact that more than half of the examined strains had the wild-type cyp51A gene supports the idea that other mechanisms of resistance are gradually increasing.

Mortality-associated factors of candidemia: a multi-center prospective cohort in Turkey.

Candidemia may present as severe and life-threatening infections and is associated with a high mortality rate. This study aimed to evaluate the risk factors associated with 30-day mortality in patients with candidemia. A multi-center prospective observational study was conducted in seven university hospitals in six provinces in the western part of Turkey. Patient data were collected with a structured form between January 2018 and April 2019. In total, 425 episodes of candidemia were observed during the study period. Two hundred forty-one patients died within 30 days, and the 30-day crude mortality rate was 56.7%. Multivariable analysis found that SOFA score (OR: 1.28, CI: 1.154-1.420, p < 0.001), parenteral nutrition (OR: 3.9, CI: 1.752-8.810, p = 0.001), previous antibacterial treatment (OR: 9.32, CI: 1.634-53.744, p = 0.012), newly developed renal failure after candidemia (OR: 2.7, CI: 1.079-6.761, p = 0.034), and newly developed thrombocytopenia after candidemia (OR: 2.6, CI: 1. 057-6.439, p = 0.038) were significantly associated with 30-day mortality. Central venous catheter removal was the only factor protective against mortality (OR: 0.34, CI:0.147-0.768, p = 0.010) in multivariable analysis. Candidemia mortality is high in patients with high SOFA scores, those receiving TPN therapy, and those who previously received antibacterial therapy. Renal failure and thrombocytopenia developing after candidemia should be followed carefully in patients. Antifungal therapy and removing the central venous catheter are essential in the management of candidemia.

Effectiveness of FastFung agar in the isolation of Malassezia furfur from skin samples.

BACKGROUND: Lipophilic basidiomycetous yeasts of the Malassezia genus can cause various skin diseases, such as seborrheic dermatitis, pityriasis versicolor, folliculitis and atopic dermatitis, and even life-threatening fungemia in newborns and immunocompromised individuals. Routine mycological media used in clinical practice do not contain sufficient lipid ingredients required for the growth of Malassezia species. A recently developed medium, FastFung agar, is promising for culturing fastidious fungal species. METHODS: In this study, we compared FastFung agar and mDixon agar for culturing Malassezia species from nasolabial fold and retroauricular specimens of 83 healthy individuals and 187 and 57 patients with acne vulgaris and seborrheic dermatitis, respectively. RESULTS: Malassezia species were identified using conventional tests and matrix-assisted laser desorption/ionisation mass spectrometry. In total, 96 of 654 samples (14.6%) contained Malassezia species. The total isolation rate was significantly higher in patients with seborrheic dermatitis (40.4%) than in healthy volunteers (21.7%; p < .05), and the rate of M. furfur isolation was significantly higher for patients with acne vulgaris (13.9%) and seborrheic dermatitis (24.6%) than for healthy individuals (1.5%; p < .05). FastFung agar was superior to mDixon agar in M. furfur isolation (p = .004) but showed similar performance in the case of non-M. furfur species (p > .05). Among cultured Malassezia species, perfect agreement between mDixon agar and FastFung agar was found only for M. globosa (κ = 0.90). CONCLUSION: Our results indicate that FastFung agar favours the growth of Malassezia species and should be useful in clinical mycology laboratories.

[Flow Cytometry Optimization of Three Different BODIPY Molecules for Staining Lipid Droplets of Cryptococcus neoformans]. / Cryptococcus neoformans'in Lipit Damlaciklarinin Boyanmasinda Üç Farkli BODIPY Bilesiginin Akan Hücre Ölçer için Optimizasyonu.

The pathogenic Cryptococcus neoformans causes life-threatening disease in immunocompromised patients. Although there are hypotheses about the role of lipid droplets in C.neoformans pathogenesis, there is still not extensively data determined on the subject yet. Lipid droplets are dynamic cytoplasmic energy storage bodies in yeasts. Diazo dyes, Nile red, LD540 and borradiazaindasen (BODIPY) molecules are frequently used in the lipid droplets studies. BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyestuffs are among the brightest green light emitting fluorophores. These neutral molecules have high lipophilicity and can easily pass through the cell wall and membrane. In this study, for the future studies on lipid droplets of C.neoformans, we aimed to optimize three different BODIPY (BODIPY480/525, BODIPY480/530, BODIPY480/535) molecules for fluorescence microscopy and flow cytometry system. Ten molecularly confirmed environmental C.neoformans strains were grown on Sabouraud dextrose agar with and without oleic acid. BODIPY staining protocols at different concentrations were used for C.neoformans lipid droplets fixed with paraformaldehyde. The visualization (by fluorescence microscopy) and detection (by flow cytometer) of the lipid droplet structures of C.neoformans strains were evaluated. Forwardscatter and side-scatter analysis were performed to evaluate the number of lipid droplets determined in the cytoplasmic region quadrant in flow cytometry. The staining of the lipid droplets of C.neoformans of all three BODIPY molecules used in the study was observed by fluorescence microscope with creating distinct brightness and sharp contrast with the background. All BODIPY molecules could be examined by fluorescence microscopy without loss of brightness in more than one minute. The optimal dye concentration of BODIPY compounds were found as 2 µM. Incubation at room temperature for five minutes was sufficient for fluorochrome staining. They were also shown to be able to stain lipid droplets in heatinactivated C.neoformans strains in all three compounds. The synthesized BODIPY480/525, BODIPY480/530 and BODIPY480/535 molecules were evaluated in accordance with the staining of lipid droplets, which were claimed to play a role in the pathogenesis of C.neoformans, and analysis by fluorescence microscopy and analysis with flow cytometer. BODIPY molecules may exhibit different properties in staining lipid droplets. These molecules should be tested for demonstrating the presence of lipid droplets in different yeast species and their suitability for pathogenesis studies.

Genotypes and population genetics of cryptococcus neoformans and cryptococcus gattii species complexes in Europe and the mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.

[Prediction of the species distribution of Cryptococcus neoformans throughout Turkey]. / Türkiye genelinde Cryptococcus neoformans'in tür dagiliminin tahmini.

Cryptococcus neoformans is a human pathogenic yeast that causes life-threatening infections especially in immunosuppressed patients. The environmental isolation of C.neoformans from Turkey was reported as early as 2004, although this was mostly from Eucalyptus camaldulensis colonization. Successful isolations were also reported from pomegranate (Punica granatum), oriental plane (Platanus orientalis), pine tree (Pinaceae), chestnut (Castanea sativa) and salt cedar (Tamarix hispida). The investigation of the relationship between the bioclimatic factors affecting the environmental isolation sites and the colonization of pathogens is a frequently used method. With this method, detailed risk maps can be generated in which environmental colonization can be estimated. The aim of this study was to use the high-resolution bioclimatic and previously-isolated yeasts' coordinates to create a valid model for the occurrence of C.neoformans in Turkey and provide insight into ecological processes. A machine learning approach using presence-only data software, maximum entropy (MaxEnt), was used to for the prediction of C.neoformans distribution. Climatic data and environmental bioclimatic variables from WorldClim were downloaded as 30 seconds spatial resolutions. The correlation between different Turkey bioclimatic layers were analyzed with ENMTools and similar layers were discarded. Forty-one different coordinates representing C.neoformans isolation points were used to generate a predictive map. The area under the curve and the omission rate were used to validate the model. Meanwhile, Jackknife tests were applied to enumerate the contribution of different environmental variables, and then to predict the final model. Maps were created using QGIS mapping software. In this study, we have shown that the coastal region of Anatolia, which is geographically located in the Northeastern Mediterranean Basin, as well as the entire Aegean region, carry an extremely high risk for the colonization of C.neoformans. Other areas which have not previously been reported for the isolation of C.neoformans were predicted to be potential colonization hotspots, including the western part of Ataturk Dam, the Amik Plain and the Bakirçay and Gediz valleys. The maximum temperature of the warmest month, the mean temperature of the warmest quarter and the precipitation of the coldest quarter were the most important factors influencing the model's predictions. It was determined that the humidity in the environment affected the colonization especially in November. In conclusion, we produced a C.neoformans colonization risk map of Turkey for the first time. Obtaining more regional data will facilitate the identification of the regions having similar risk. This approach is useful for the clinical prediagnosis of cryptococcosis cases, which may be more common in places with environmental niches.

[Isolation of Cryptococcus neoformans from a chesnut tree (Castanea sativa), Denizli, Turkey]. / Denizli bölgesinde kestane (Castanea sativa) agacindan Cryptococcus neoformans izolasyonu.

Cryptococcus neoformans is a basidiomycetous encapsulated yeast that can cause life-threatening infections in immunosuppressed humans and animals. C.neoformans/Cryptococcus gattii infections are considered to be acquired via inhalation of aerosolized particles from the environment. Avian guano, decaying tree hollows and soil are known as environmental niches. In recent years, colonization of the woody structures of different trees such as Eucalyptus camaldulensis, Tamarix hispida, Platanus orientalis and Punica granatum has been reported in the environmental study of the western Anatolian region. Based on the results of previous studies, our country may have intensive Cryptococcus colonization niches in the western regions. The aim of this study was to investigate the presence of the colonization of C.neoformans niche in chestnut (Castanea spp.) trees on higher altitudes. In the study, the colonization of C.neoformans was screened on chestnut trees (Castanea spp.) in Aydin-Ödemis-Denizli geographical area. This area consists of mountainous terrain between the fertile plain formed by two major rivers.This region is one of the widespreading areas of chestnut farming in Anatolia. Two hundred and fourteen chestnut trees that had deep fissures or trunk hollows were screened during mid-summer 2017. A swabbing technique was used, and all samples were cultured on Staib agar medium containing biphenyl and antibiotics. Cultures were checked for ten days for suspicious brown colonies. Suspicious yeast colonies were tested for the identification of pathogenic Cryptococcus by conventional methods and canavanine-glycine-bromothymol agar reactions. ITS 1-4 primers were used for strain PCR tests. We determined the mating type and serotypes by PCR analysis of the STE20 genes using STE20 (Aa), STE20 (Aα), STE20 (Da), and STE20 (Dα) primers. V8 agar medium was used for mating cultivation. Only one (0.47%) strain of C.neoformans was isolated from 214 screened trees. This strain was confirmed by ITS 1-4 sequencing. The serotype A MATα mating type was observed. Basidium, basidiospores and clamp connections in hyphal structure were noted with MATα mating on V8 agar medium. In this study, the first C.neoformans isolate from a chestnut tree (Castanea sativa) was determined from Denizli region. Further studies of distribution of human pathogenic Cryptococcus will be helpful to determine the risk areas for the living organisms in our region.

[A comparison of biosurfactant production between clinical and environmental Exophiala isolates]. / Klinik ve çevresel Exophiala izolatlarinda biyosurfaktan üretiminin karsilastirilmasi.

Black yeast in the genus Exophiala are able to grow in hydrocarbon-contaminated environments and are pathogenic in immunosuppressed hosts. The biosurfactant produced by Exophiala species may be associated with strain pathogenicity by changing the hydrophobicity. The aim of this study was to prove the hypothesis that biosurfactant production in Exophilia strains isolated from clinical samples is lower than the strains isolates from toxic (dishwasher and railway sleepers) environments. A total of 122 Exophiala isolates 108 environmental (isolated from 82 dishwashers and 36 railway sleepers) and 14 clinical isolates confirmed by molecular tests were included in the study. Biosurfactant activity was tested by the drop collapse method, in which the surface of a microtiter plate well was evaluated for the presence of a biosurfactant, and by the oil spreading technique on crude oil. An open source analyses program, ImageJ®, was used for crude oil spreading technique data. A clear surface zone that differs more than two standard deviations from the mean size was accepted as a positive result. Among the 122 Exophiala species, 11 (9.0%) and 10 (8.2%) strains showed biosurfactant activity by the drop collapse test and oil spreading method, respectively. An acceptable relation was found between the drop collapse test and oil spreading method (Cohen κ coefficient= 0.30). Despite the presence of isolates showing biosurfactant activity, no statistically significant difference was detected between Exophiala strains (p= 0.72). The biosurfactant levels of environmental isolates were higher than the isolates obtained from the patients (p= 0.03). The highest biosurfactant level was observed in one Exophiala phaeomuriformis strain isolated from a dishwasher. There was no difference between the biosurfactant levels of the dishwasher and railway sleeper isolates (p= 0.66). Biosurfactant production may be a more important determinant of virulence in Exophiala species than expected. In this study, biosurfactant activity was higher in environmental isolates compared to the clinical isolates. Consensus of multiple biosurfactant screening protocols may clarify why environmental Exophiala species are less virulent. Further studies should evaluate biosurfactant activity in additional clinical Exophiala isolates. The biosurfactant activity of more Exophiala isolates obtained from patients should be investigated with further planned studies.

Fundamental niche prediction of the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii in Europe.

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe and Mediterranean area.

[A case with scalp pruritus caused by Dermanysus gallinae (order: mesostigmata)]. / Dermanysus gallinae'nin (Takim: mesostigmata) neden oldugu saçli deride kasintili olgu.

Dermanyssus gallinae is one of the important hematophagous ectoparasite species of poultry like chicken, pigeon and wild bird species. These ectoparasites in the form of nymphs or adults who can not find their hosts are also seen in mammals and even in humans. For this reason, they are considered as important for public health. The ectoparasite causes a clinical condition named gamasoidosis among pet owners and people who live or work close to animal shelters, barns and chicken farms. Pruritus dermatitis is also caused by D.gallinae in humans and can cause false diagnosis. In this report, a case of D.gallinae which leads to severe itch in the hairy head skin was presented. A 66-year-old female patient admitted to University Hospital with complaints of "bugs in her hair and itching of the skin increasing in the evenings" that have persisted for a month. In the dermatological examination of the patient, it was noted that her hair and scalp were usual. Routine laboratory tests were normal. However, a large number of mites were found in her headscarf that she brought with her to the examination. Later, it was learned that the patient feeds chicken in her garden in the village where she lives. The collected mite samples were were kept in glass test tubes that contained glycerol and alcohol. The mites were identified as D.gallinae by morphological identification with light microscopy by using 10x, 20x and 40x magnifications. The mites were described as D.gallinae (Order: Mesostigmata, local name: poultry red mite, perch mite, poultry mite) with the morphological examination. Long-acting 1% permethrin shampoo was applied to remove the mites on the patient and during the controls, it was changed as 5% permethrin and 10% crotamiton lotion. For environmental sanitation, carbamates (such as carbolineum, trichlorfon, malathion, tetrachlorvinphos, etc.), organophosphates and acaricide insecticides with pyrethroids spraying or powder formulations were recommended. It was recommended to repair the slits and cracks where the parasite in the shelter could be stored. The patient was informed on (i) how to clean the household items with susceptible acaricides, (ii) removal of unused infected animal shelters, cages and nests from human habitat, (iii) raising of ambient temperature above 45°C, (iv) ventilation of the living spaces and (v) washing the clothes with detergent. In order to be effectively protected from the risk of infection and the detriments that are brought by this parasite, it is imperative to stay away from the hosts and the infected areas such as chicken farms, to obey the hygiene regulations, and to properly conduct the disinfestation of the shelters. In addition, it is also helpful to receive a true story from the patient, with details of contact with birds for the protection and treatment.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

ATR-FTIR Spectroscopy Highlights the Problem of Distinguishing Between Exophiala dermatitidis and E. phaeomuriformis Using MALDI-TOF MS.

The present study compared two chemical-based methods, namely, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, to understand the misidentification of Exophiala dermatitidis and Exophiala phaeomuriformis. The study utilized 44 E. dermatitidis and 26 E. phaeomuriformis strains, which were partially treated with strong acids and bases for further evaluation. MALDI-TOF MS and ATR-FTIR spectroscopy data of the two Exophiala species were compared. Data groupings were observed for the chromic acid- and nitric acid-treated species when the black yeast sources were categorized as creosoted-oak sleepers, concrete sleepers, or dishwasher isolates. The MALDI-TOF MS data for the metalloenzyme-containing regions were consistent with the ATR-FTIR spectroscopy data. These results indicated that environmental isolates might contain metals not found in human isolates and might interfere with chemical-based identification methods. Therefore, MALDI-TOF MS reference libraries should be created for clinical strains and should exclude petroleum-associated environmental isolates.

Pulmonary mucormycosis due to Lichtheimia ramosa in a patient with HIV infection.

Mucormycosis is increasingly common in patients with risk factors such as diabetes mellitus, neutropenia, and corticosteroid therapy. However, mucormycosis seems to be less common in patients with human immunodeficiency virus (HIV) infection compared to patients with other risk factors. Despite their lower virulence, Lichtheimia species should be regarded as emerging pathogens among Mucoralean fungi. We report a fatal case of pulmonary mucormycosis due to Lichtheimia ramosa in a 52-year-old man with an end-stage HIV infection. He had a cachectic appearance and his CD4 count was 8 cells/mm(3). The fungal infection was diagnosed based on a positive sputum culture with histopathologic confirmation. The fungus was resistant to caspofungin, anidulafungin, and voriconazole [minimum inhibitory concentration (MCI) >32 µg/ml], whereas the E test MIC values of itraconazole, posaconazole, and amphotericin B were 0.38, 0.38, and 0.5 µg/ml, respectively. Although intravenous drug use is the main risk factor for the development of mucormycosis in HIV-infected patients, it may also develop in patients with low CD4 count, opportunistic infections and/or additional diseases, such as Kaposi's sarcoma or severe immunodeficiency, as in our case.

[Evaluation of a new medium, eggplant (Solanum melongena) agar as a screening medium for Cryptococcus neoformans in environmental samples]. / Çevresel Örneklerde Cryptococcus neoformans Taramalari Için Patlican (Solanum melongena) Agarin Yeni Bir Besiyeri Olarak Degerlendirilmesi.

Cryptococcus neofomans is an encapsulated yeast-like fungus that causes life-threatening infections, especially in immunosuppresive patients. C.neoformans infection is believed to be acquired via inhalation of aerosolized particles from the environment. Avian guano, decaying tree hollows and soil are the related known environmental niches. Brown pigmented yeast growth from the precursors in growth media is an important step for the identification and isolation of C.neoformans. Seeds of plants in nature are preferred owing to easy accessibility and low costs for the preparation of such media. Guizotia abysinicca (Niger seed) as Staib agar, Helianthus annus (Sunflower) as Pal's medium, Brassica nigra (Mustard) agar, tobacco agar, Mucuna pruriens (Velvet bean) seed agar, Perilla frutescens (Beefsteak plant) seed agar, Rubus fruticosus (Blackberry) agar and ground red hot pepper agar are pigment-based selective media for the differentiation of C.neoformans. The aim of this study was to observe the pigment production of C.neoformans in a new medium based on eggplant (Solanum melongena) and also to compare its performance with the simplified Staib, Pal's and tobacco agar for isolation from the environment. Three different eggplant-based medium (S.melongena Melanzaza viserba, S.melongena Pinstripe F1 and S.ovigerum Ivory F1) were included in the study. Pigment-forming eggplant medium, simplified Staib agar, Pal's agar and tobacco agar were used for the cultivation of the environmental swabbed samples from 19 Eucalyptus camaldulensis trunk hollows in continuous colonization region. While pigment formation were observed with S.melongena Melanzaza viserba and S.melongena Pinstripe F1 containing media, S.ovigerum Ivory F1 medium was found to be non-reactive. In colonization area (Gökova-Akyaka, Turkey), 11 (57.9%) out of 19 E.camaldulensis samples were positive with simplified Staib agar, Pal's agar and eggplant agar while 10 (52.6%) of them are positive with tobacco agar. C.neoformans colony forming unit (cfu) per plate were found as 51, 57 and 48 (median values) on simplified Staib agar, Pal's agar and eggplant agar, respectively, while tobacco agar has lower performance with 33 cfu/petri. No statistically significant difference were found between simplified Staib agar, Pal's agar and eggplant agar's performances for C.neoformans isolations from the nature (p=0.71). In conclusion, easily prepared eggplant agar is as functional as widely used media such as simplified Staib agar and Pal's agar for the isolation of C.neoformans from the natural environment.

[The effect of Tween-80 on the differentiation of Trichophyton mentagrophytes and Trichophyton rubrum strains with FT-IR spectroscopy]. / Trichophyton mentagrophytes ve Trichophyton rubrum kökenlerinin FT-IR spektroskopi ile ayriminda Tween-80 etkisi.

Trichophyton mentagrophytes and Trichophyton rubrum, are two of the frequently identified dermatophyte species in routine microbiology laboratories. Although newer technologies may assist in species-level identification, direct application of these methods usually require improvement in order to obtain reliable identification of these species. Earlier data have shown that dermatophytes may be identified with FT-IR spectroscopy although there are some limitations. In particular, the organic bond ranges in FT-IR spectra showed more irregularity because of the eucaryotic complexity of the molds. In this study, Tween-80 which is an inorganic molecule, was added to the dermatophyte growth medium in order to investigate its effect on FT-IR spectroscopy analysis of dermatophytes. Nine reference dermatophyte strains [5 T.mentagrophytes complex (T.asteroides CBS 424.63, T.erinacei CBS 344.79, CBS 511.73, CBS 677.86, T.mentagrophytes CBS 110.65) and 4 T.rubrum complex strains with different morphotypes (T.fluviomuniense CBS 592.68, T.kuryangei CBS 422.67, T.raubitschekii CBS 102856, T.rubrum CBS 392.58)] were included in the study. All strains were cultured on Sabouraud glucose agar either with or without 1% Tween-80 for three weeks. After the incubation period, superficial scrapings from each dermatophyte colony were analyzed using FT-IR spectroscopy. All measurements were performed in transmission mode between 4400 and 400 cm-1. Numerous spectral window data were analyzed by principal component analysis and hierarchical clustering was performed. The second derivations of spectral ranges revealed clear grouping of T.mentagrophytes complex and T.rubrum complex in association over five separate spectral ranges. The findings also showed that while all of the T.mentagrophytes strains contained lipid compounds in their mold structure after Tween-80 incubation (p< 0.025), T.rubrum strains did not. Based on these results, it was concluded that culture medium containing Tween-80 was sufficient to enable differentiation of T.mentagrophytes complex from T.rubrum complex by FT-IR spectroscopy. This effect might be attributed to the possible transfer of lipid compounds from culture to cell structure during growth. Further studies with the use of large number of reference strains and clinical isolates exposed to different environmental factors, such as antifungal agents and inorganic ions, are needed to support these data indicating favorable effect of Tween-80 on the differentiation of T.mentagrophytes and T.rubrum complexes by FT-IR spectroscopy.

Evaluation of novel cosmetic shampoo formulations against Malassezia species: Preliminary results of anti-dandruff shampoo formulations.

OBJECTIVES: Malassezia species are common, clinically relevant, and lipid-dependent yeasts of humans. They are also the leading causes of the dandruff problem of humans, and the azoles are used primarily in their topical and systemic treatment. Resistance to azoles is an emerging problem among Malassezia sp., which indicates the need of new drug assessments that will be effective against dandruff and limit the use of azoles and other agents in treatment. Among them, the efficacy of various combinations of piroctone olamine and climbazole against Malassezia sp. is highly important. Here, we assessed the efficacies of various piroctone olamine and climbazole formulations against Malassezia sp. in comparison with ketoconazole. METHODS: A total of nine formulations were included in the study, where each formulation was prepared from different concentrations of piroctone olamine and climbazole and both. All formulations contained the same ingredients as water, surfactants, hair conditioning agents, and preservatives. Malassezia furfur CBS1878, Malassezia globosa CBS7874, and Malassezia sympodialis CBS9570 were tested for antifungal susceptibility of each formulation by agar diffusion method. Sizes of the inhibition zones were compared with standard medical shampoo containing 2% ketoconazole, and the data were analyzed by Dunnett's multiple-comparison test. RESULTS: For all Malassezia sp. strains, climbazole 0.5% and piroctone olamine/climbazole (0.1%/0.1% and 0.1%/0.5%) combinations were found to have the same effect as the medical shampoo containing 2% ketoconazole. Piroctone olamine/climbazole 1.0%/0.1% formulation showed the same efficacy as 2% ketoconazole on M. furfur and M. sympodialis, while 0.1%/0.5% formulation to only M. furfur. For M. globosa, none of the formulations tested were as effective as ketoconazole. CONCLUSION: The species distribution of Malassezia sp. varies depending on the anatomical location on the host. According to the results of this study, climbazole and piroctone olamine combinations seem to be promising options against the dandruff problem with their high antifungal/anti dandruff efficacy.