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.

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.

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.

Microbiological analysis of a mummy from the archeological museum in Zagreb.

In this paper we report the results of the microbiological analysis of the samples taken from the mummy from the collection of the Archaeological museum in Zagreb, Croatia. Samples were taken from specific places such as oral, orbital, abdominal cavity and bandages surrounding the mummy, and analyzed in Department of Microbiology and Hospital Infections in University Hospital "Dubrava" in Zagreb and in National Reference Laboratory for systemic mycoses of Croatian National Institute of Public Health in Zagreb. The analysis indicated that all of the found organisms were non-primary pathogenic and are not harmful for healthy humans. Isolated microorganisms mainly belonged to the group of saprophytic fungi as listed: Monilia spp., Penicillium spp., Alternaria spp., Aspergillus fumigatus, Aspergillus nidulans, Rhizopus spp. and Chrysosporium spp. and to the genus of saprophytic bacteria, Bacillus spp.

First Isolation, Antifungal Susceptibility, and Molecular Characterization of Cryptococcus neoformans from the Environment in Croatia.

The purpose of this study was to investigate the presence of Cryptococcus neoformans species complex isolates from environmental sources in Croatia and to determine their molecular types and antifungal susceptibility. Swab samples of tree hollows and bird excreta in the soil beneath trees were collected. Samples included 472 (92.73%) samples obtained from tree hollows and 37 (7.27%) samples from bird excreta. Four C. neoformans species complex isolates were recovered from tree hollow swabs along the Mediterranean coast, while there were no isolates recovered from bird excreta or from the continental area. Three isolates were identified as molecular types VNI and one as VNIV. All tested antifungals showed high in vitro activity against the four isolates. This is the first report proving the presence of C. neoformans species complex in the environment of Croatia. The results of the study suggest a major risk of exposure for inhabitants living along the Croatian coast and that both VNI and VNIV molecular types can be expected in clinical cases of cryptococcosis. Susceptibility to antifungals confirmed that no resistance should be expected in patients with cryptococcosis at the present time.

Agricultural systems as potential sources of emerging human mycoses caused by Trichoderma: a successful, common phylotype of Trichoderma longibrachiatum in the frontline.

Trichoderma species are abundant in different agricultural habitats, but some representatives of this genus, mainly clade Longibrachiatum members are also emerging as causative agents of various human diseases with even fatal outcome. Strains of these species frequently show resistance to commonly used azole antifungals. Based on previous data it is hypothesized that Trichoderma isolates identified in human infections derive from environmental-including agricultural-origins. We examined Trichoderma longibrachiatum Rifai and Trichoderma bissettii Sandoval-Denis & Guarro strains recovered from four novel cases of human mycoses, along with isolates from previous case reports and different agricultural habitats, using multilocus phylogenetic analysis, BIOLOG Phenotype Microarrays and Etest. Strains attributed to T. bissettii were more abundant in both clinical and agricultural specimens compared to T. longibrachiatum. The majority of the isolates of both taxa could tolerate >256, >32 and >32 µg/ml fluconazole, itraconazole and posaconazole, respectively. None of the obtained results revealed characteristic differences between strains of clinical and agricultural origin, nor between the two taxa, supporting that agricultural environments may be significant sources of infections caused by these emerging human fungal pathogens. Furthermore, based on our findings we propose the re-classification of T. bissettii as T. longibrachiatum f. sp. bissettii.

[Proteinase excretion ability of Candida albicans species isolates in clinical specimens]. / Ispitivanje sposobnosti lucenja proteinaze izolata vrste Candida albicans izdvojenih iz klinickih uzoraka.

AIM: The aim of the study was to establish whether the Candida (C.) albicans species isolates from samples of the patient digestive, respiratory and genitourinary systems differed in the ability of proteinase excretion. METHODS: A total of 1009 isolates of the C. albicans species obtained from 1009 clinical specimens of the digestive, respiratory and genitourinary systems of 666 patients were examined. All samples were inoculated onto Sabourauds glucose agar and incubated aerobically at 37 degrees C for 3-7 days. Identification of C. albicans was done by standard and commercial tests. To test the proteinase excretion ability, we employed the Odds and Abbott method. The isolates were inoculated on a culture medium containing agar, glucose, vitamins and beef albumin fraction V (pH = 3.2), and stored for 7 days at 30 degrees C. Each isolate was tested twice, with the results read by the same person. Development of thready colonies in a milky-white field was considered as positive finding. Study results are shown in easy-to-consult tables. Distribution differences were assessed by chi2-test. RESULTS: Of 1009 C. albicans species isolates obtained from clinical specimens, 695 (68.9%) had the proteinase excreting ability. The presence of this enzyme was demonstrated in 72.7% of the species isolates obtained from samples of the digestive system, 65.8% of isolates from respiratory system and 59.6% of isolates from genitourinary system. Analysis of chi2-test results showed no statistically significant difference in the ability of C. albicans species isolates from specimens obtained from digestive, respiratory and genitourinary systems to excrete proteinase. DISCUSSION: The results of the present study are in agreement with the results of most other researchers reporting on the proteinase excreting ability to be demonstrated in 40%-80% of C. albicans isolates. In the present study, the ability to excrete proteinase was demonstrated in 68.9% of C. albicans species isolates obtained from clinical samples. CONCLUSION: The ability to excrete proteinase was demonstrated in 59.6%-72.7% of C. albicans species isolates obtained from the patient digestive, respiratory and genitourinary systems. Analysis of the results yielded no statistically significant difference in the proteinase excreting ability among the isolates obtained from digestive, respiratory and genitourinary systems.

Biotypes of Candida albicans isolated from cardiovascular system and skin surveillance cultures of hospitalized patients.

The aim of the study was to biotype 59 isolates of Candida (C.) albicans from cardiovascular system samples (blood and intravenous catheter) and 123 isolates of the same species from skin surveillance cultures (swabs of the armpit, groins and intravenous catheter insertion sites) of hospitalized patients using the Odds and Abbott biotyping method. Biotyping of 59 isolates of C. albicans taken from the cardiovascular system samples revealed the presence of 16 biotypes. Biotype 355 was the most common biotype, accounting for 35.6% of all biotype isolates from this system. Biotyping of 123 C. albicans isolates from skin surveillance cultures detected 21 biotypes. Biotype 355 was most common, accounting for 17.9% of all biotype isolates from these samples. The two systems had 10 biotypes in common: 355, 155, 257, 305, 105, 315, 300, 015, 157, and 345. These biotypes accounted for 88.3% and 81.4% of all C. albicans biotypes isolated from the cardiovascular system and skin surveillance cultures, respectively. Biotypes 355, 155, and 257 were the biotypes most frequently shared in isolates from the two systems. These biotypes accounted for 57.7% and 43.1% of all C. albicans biotypes isolated from the cardiovascular system and skin surveillance cultures, respectively.

Species distribution and frequency of isolation of yeasts and dermatophytes from toe webs of diabetic patients.

The paper identifies fungal species, looking at the incidence of fungal isolation and risk factors influencing the development of fungal infection and colonization of interdigital spaces of the feet in 509 diabetic outpatients. Using standard mycologic diagnostic methods, fungi were detected in toe webs of 122 (24%) diabetic patients. The finding of fungi was twice as common in interdigital spaces of one (85/16.7% of the patients) than both feet (37/7.3% of the patients). Yeasts were the most common isolates (95/18.7% of the patients), followed by dermatophytic moulds (24/4.7% of the patients), whereas coexistence of yeasts and dermatophytes was the most infrequent finding (3/0.6% of the patients). From toe webs, 24 fungal species, 21 yeast species belonging to nine genera (Candida, Rhodotorula, Cryptococcus, Trichosporon, Saccharomyces, Blastoschizomyces, Geotrichum, Debaryomyces, and Ustilago) as well as three species of dermatophytes of the genera Trichophyton and Epidermophyton were isolated. The most frequently isolated fungi were Candida parapsilosis (59/11.6% of the patients) and Trichophyton mentagrophytes (16/3.1% of the patients). Although there was no correlation between the incidence of toe web space colonization with yeasts and dermatophytosis with the criteria of patient sex and age, and duration of diabetes, the difference in the incidence according to type of diabetes was statistically significant. In non insulin dependent diabetes mellitus patients, the incidence of fungal isolation from toe webs was statistically significantly higher (30.1%) than in insulin dependent diabetes mellitus patients (19.8%).

Fungal infections of urogenital system.

We analyzed the frequency of isolation of individual fungal species in the samples of urine, vaginal and cervical swabs from 107 patients (72% women and 28% men) aged 16-82 years, who were treated in primary care for cystitis, vulvovaginitis, and cervicitis. The samples were analyzed at the Microbiological Laboratory of Zagreb Institute of Public Health, Croatia, between September 1, 2002 and June 31, 2003. Eight species of yeast were isolated from the samples. Candida (C.) albicans, C. glabrata, and C. krusei were the most common isolates, with the frequency of 61.7%, 10.3% and 8.4%, respectively. Other species (C. guilliermondii, C. famata, C. tropicalis, C. parapsilosis, and C. kefyr) were less frequently isolated, between 0.9% and 7.5%. In women, the frequency of isolation of C. albicans species from urine samples ranged from 83.3% to 30.8%, and from uterine cervical swabs from 85.7% to 50%, showing a decreasing trend with patients' age. The frequency of C. albicans isolates from vaginal swabs was equal in younger (<30 years) and older women (l51 years), but twice less frequent in middle-aged women. In men, the frequency of C. albicans species isolated from urine samples decreased with age from 100% to 52.4%. In the 107 analyzed samples with positive fungal culture, yeast isolations were significantly more frequent than pure culture (93.5%) and had a larger number of colony counts (57%). This suggested that these yeast species might have a pathogenic role in the causation of urogenital system infections.

Prevalence of Pneumocystis jirovecii pneumonia (2010-2013): the first Croatian report.

Pneumocystis jirovecii is an important cause of interstitial pneumonia particularly among immunocompromised hosts. We analysed the prevalence of P. jirovecii pneumonia (PCP) among HIV-infected and HIV-uninfected patients presented with interstitial pneumonia or acute respiratory syndrome hospitalized in six Croatian tertiary care hospitals. Over four-year period (2010-2013), a total of 328 lower respiratory tract samples: 253 (77.1%) bronchoalveolar lavage fluid, 43 (13.1%) tracheal aspirates and 32 (9.8%) bronchial aspirates from 290 patients were examined by real-time polymerase chain reaction (PCR). PCP was detected in 23 (7.9%) patients. The prevalence of PCP differed significantly among tested groups (χ2 = 95.03; d.f. = 3; p < 0.001). HIV-infected patients were more often positive (56.6%, 95%CI = 37.3-72.4) compared to other groups (patients with malignant disease 7.7%, 95%CI = 2.6-20.3; transplant patients 7.7%, 95%CI = 2.2-24.1; patients with other diagnosis 1.5%, 95%CI = 0.5-4.4). Majority of HIV-positive patients (80%) were newly diagnosed cases. Our results indicate that HIV-infected patients still represents the main risk group for P. jirovecii infection. PCP is responsible for pneumonia in 56.6% HIV-positive patients in Croatia, primarily those who do not know that they are HIV infected.

The use of paleo-imaging and microbiological testing in the analysis of antique cultural material: multislice tomography, and microbial analysis of the Trogir Cathedral cope hood depicting St. Martin and a beggar.

Paleoradiology is the study of biological and other materials from archeological settings through the use of various medical imaging techniques. Although it is most often used in the scientific study of ancient human remains, it can also be used to study metals, ceramics, paper, and clothes. The aim of this study was to test two paleoimaging techniques (MSCT and mammography) in the analysis of an important Croatian liturgical vestment: the hood of a bishop's cope from St. Lawrence's Treasury in Trogir depicting St. Martin and a beggar. To ensure a safe environment for scientists participating in the analysis, a preliminary microbiological analysis was performed, which contributed to the database of microbiological flora found on Croatian archeological remains and relics studied to date. Due to a great amount of metal filaments, the paleoradiological analysis did not produce satisfactory results. However, a digitally enhanced image clearly showed fine metal embroidery of the hood that was not so easily perceived by naked eye. This article argues in favor of expanding paleoradiological studies on materials other than human remains and also of publishing unsatisfactory results, as important lessons for future development of techniques and methods to analyze ancient remains and seek answers about human historical and cultural heritage.

Two rare cases of central nervous system opportunistic mycoses.

This article presents two cases of opportunistic mycoses (OMs) of the central nervous system (CNS) caused by Cryptococcus neoformans and Aspergillus nidulans, respectively. The patients were hospitalised in local hospitals between 2009 and 2011 because of unspecific symptoms (fever, headache, and/or weight lost). Duration of symptoms varied from 4 days to over 2 weeks. The patients were treated with antibiotics and symptomatically. OM was not suspected in any of them. The patients became critically ill with symptoms of CNS involvement and were transferred to the Intensive Care Unit (ICU) of the University Hospital for Infectious diseases (UHID) in Zagreb. None of the patients belonged to the high-risk population for developing OMs. They were not HIV-infected, had no transplantation of bone marrow or solid organ, and were not on severe immunosuppressive chemotherapy. Fungi were isolated from cerebrospinal fluid (CSF) samples and, in one patient, from aspirate of cerebral abscess. Isolation and mycological identification of all fungal isolates and in vitro antifungal susceptibility testing of these isolates were done at the Reference Centre for Mycological Diagnostics of Systemic and Disseminated Infections (RCMDSDI) in Zagreb. The patient with cryptococcal meningitis was treated with amphotericin B and fluconazole and the patient with cerebral aspergilloma with voriconazole.

Chronic fungal meningitis caused by Aureobasidium proteae.

We present a case of chronic meningitis due to the mold Aureobasidium proteae. Clinical features, the disease course, as well as the diagnostic methods and optimal treatment options are discussed. This case confirms the neuroinvasiveness of A. proteae and introduces it as a new human pathogen.

Candida utilis candidaemia in neonatal patients.

In recent years, an evident rise in the frequency of candidaemia caused by non-albicans Candida species has been reported. In this paper we present three cases of clinically manifested candidaemia caused by Candida utilis in neonatal patients hospitalized in the same neonatal intensive care unit within a 6 month period. To the authors' knowledge, only two cases of C. utilis candidaemia have been reported in the literature to date, but neither of these involved newborns. Clinical resolution and elimination of C. utilis from the blood were achieved using liposomal amphotericin B or caspofungin in all patients.

In vitro antifungal susceptibilities and molecular typing of sequentially isolated clinical Cryptococcus neoformans strains from Croatia.

A collection of 48 clinical Cryptococcus neoformans isolates from Croatia was investigated retrospectively using in vitro antifungal susceptibility testing and molecular biological techniques to determine mating type and serotype by PCR and amplified fragment length polymorphism (AFLP) genotyping. These isolates were obtained from 15 patients: ten were human immunodeficiency virus (HIV)-negative (66.7 %) and five were HIV-positive (33.3 %). From five patients, only one isolate was available, whilst from the other ten patients, two to 11 isolates were isolated sequentially. Antifungal susceptibility was tested by a broth microdilution method. Serotype A (genotype AFLP1) and serotype D (genotype AFLP2) were both found in six patients (40 % each), and serotype AD (genotype AFLP3) in three (20.0 %) patients. Mating type α (n = 12; 80.0 %) predominated and α/a hybrids were identified in 20.0 % of patients diagnosed with cryptococcosis. Two AFLP genotypes of C. neoformans were isolated during a single episode from one patient. The in vitro antifungal MIC(90) and susceptibility ranges for C. neoformans isolates were 0.5 µg ml(-1) (range 0.031-0.5 µg ml(-1)) for amphotericin B, 4 µg ml(-1) (range 1-4 µg ml(-1)) for flucytosine and fluconazole, 0.25 µg ml(-1) (range 0.031-0.5 µg ml(-1)) for itraconazole and 0.062 µg ml(-1) (range 0.031-0.25 µg ml(-1)) for voriconazole.