Virulence Factors and in-Host Selection on Phenotypes in Infectious Probiotic Yeast Isolates (Saccharomyces 'boulardii').

Saccharomyces yeast probiotics (S. 'boulardii') have long been applied in the treatment of several gastrointestinal conditions. Despite their widespread use, they are rare opportunistic pathogens responsible for a high proportion of Saccharomyces mycosis cases. The potential virulence attributes of S. 'boulardii' as well as its interactions with the human immune system have been studied, however, no information is available on how these yeasts may change due to in-host evolution. To fill this gap, we compared the general phenotypic characteristics, cell morphology, virulence factors, epithelial and immunological interactions, and pathogenicity of four probiotic product samples, two mycosis, and eight non-mycosis samples of S. 'boulardii'. We assessed the characteristics related to major steps of yeast infections. Mycosis and non-mycosis isolates both displayed novel characters when compared to the product isolates, but in the case of most virulence factors and in pathogenicity, differences were negligible or, surprisingly, the yeasts from products showed elevated levels. No isolates inflicted considerable damage to the epithelial model or bore the hallmarks of immune evasion. Our results show that strains in probiotic products possess characteristics that enable them to act as pathogens upon permissive conditions, and their entry into the bloodstream is not due to active mechanisms but depends on the host. Survival in the host is dependent on yeast phenotypic characteristics which may change in many ways once they start evolving in the host. These facts call attention to the shortcomings of virulence phenotyping in yeast research, and the need for a more thorough assessment of probiotic use.

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.

Epidemiologic characteristics of Helicobacter pylori infection in southeast Hungary.

BACKGROUND: Epidemiologic studies have revealed a decrease in the prevalence of Helicobacter pylori (H. pylori) infection in Western Europe. AIM: To obtain data regarding the prevalence of H. pylori in Csongrád and Békés Counties in Hungary, evaluate the differences in its prevalence between urban and rural areas, and establish factors associated with positive seroprevalence. METHODS: One-thousand and one healthy blood donors [male/female: 501/500, mean age: 40 (19-65) years] were enrolled in this study. Subjects were tested for H. pylori IgG antibody positivity via enzyme-linked immunosorbent assay. Subgroup analysis by age, gender, smoking habits, alcohol consumption, and urban vs non-urban residence was also performed. RESULTS: The overall seropositivity of H. pylori was 32%. It was higher in males (34.93% vs 29.2%, P = 0.0521) and in rural areas (36.2% vs 27.94%, P = 0.0051). Agricultural/industrial workers were more likely to be positive for infection than office workers (38.35% vs 30.11%, P = 0.0095) and rural subjects in Békés County than those in Csongrád County (43.36% vs 33.33%, P = 0.0015). CONCLUSION: Although the prevalence of H. pylori infection decreased in recent decades in Southeast Hungary, it remains high in middle-aged rural populations. Generally accepted risk factors for H. pylori positivity appeared to be valid for the studied population.

Epidemiology of candiduria and Candida urinary tract infections in inpatients and outpatients: results from a 10-year retrospective survey.

INTRODUCTION: The presence of Candida species in urine (candiduria) is a common clinical finding, which may frequently represent colonization or contamination of specimens, however, they may be etiological agents in urinary tract infections (UTIs) or be indicators of underlying pathology in the genitourinary system or disseminated candidaemia. C. albicans is the most frequently isolated species of the genus, however, an increase in the occurrence of non-albicans Candida species (NACS) has been reported, which may be attributable to frequent exposure to fluconazole. MATERIAL AND METHODS: The aim of this study was to retrospectively assess and compare the prevalence of candiduria and UTIs caused by Candida spp. among inpatients and outpatients at a major clinical center in Hungary, during a 10-year period (2008-2017). RESULTS: Candiduria was detected in 0.11-0.75% of positive samples from outpatients, while this number was significantly higher for inpatients, ranging between 3.49-10.63% (p <0.001). Overall, C. albicans was the most frequently isolated species (65.22% in outpatients and 59.64% in inpatients), however, the presence of C. glabrata as a relevant etiologic agent (~20-30%) is also noteworthy, because there are corresponding therapeutic consequences. CONCLUSIONS: A pronounced female dominance (1.7-2.15-fold), advanced age (~70 years) and hospitalization of affected patients during candiduria is in line with the findings in literature.

Description of five dirofilariasis cases in South Hungary and review epidemiology of this disease for the country.

BACKGROUND: Dirofilariasis is an emerging zoonosis (supported by climate change) in Central Europe. Human infections are usually caused by Dirofilaria repens and Dirofilaria immitis with mediation of mosquito vectors. Aims of this publication were to report our dirofilariasis cases, and to summarize Hungarian epidemiological data by reviewing literature. METHODS AND RESULTS: We present five (four ophthalomological, one subcutaneous) cases observed within a 2-year period in Southern Hungary. Ages of infected patients were between 31 and 74 years. First case during pregnancy is also reported. There was no travel history in the anamnesis of patients which could explain acquisition of the infection. Moving, intact worms eliminated by surgical interventions were identified on the basis of morphological features as D. repens. Since the first report of human case, 115 further episodes (in addition to ours) were diagnosed in Hungary. Mean age of the patients was 47 years. Reviewing national reports, the ratio of subcutaneous infections was higher than that of the ocular ones (66 and 45, respectively). Evaluation of the territorial distribution of human episodes revealed that most infections occurred in patients living in the Danube-Tisza interfluvial region and eastern part of the country. However, sporadic cases were also found in western counties during 2014. CONCLUSION: Most of the Hungarian dirofilariasis cases were autochthonous infections. Occurrence in the western counties may suggest the spreading of this emerging zoonosis to these areas. Comprehensive monitoring and data analysis are desirable, therefore reporting the epidemiologic data in the case of human infections should be made mandatory.

Do not forget the stool examination!-cutaneous and gastrointestinal manifestations of Blastocystis sp. infection.

Blastocystis sp. is one of the most common parasites in the human intestinal tract. This infection commonly is accompanied by diarrhoea and abdominal pain, but extraintestinal symptoms, such as skin lesions, may also accompany the disease. In this study, our aim was to assess the frequency, clinical symptoms and skin manifestations of confirmed positive Blastocystis sp. infections. Data of 80 patients with confirmed positive Blastocystis sp. infections were assessed retrospectively. The average age of the patients was 46.3 years of age (with a range between 13 and 85 years of age). The number of female patients was higher than the number of males (48 vs. 32; 60 vs. 40%). Gastrointestinal and dermatological symptoms and the results of routine biochemical and haematological blood tests of enrolled patients were collected and analyzed. The skin manifestations were analyzed using the data available (including descriptions, photos and histologies). We discovered that 11.25% of our enrolled patients exhibited skin manifestations associated to Blastocystis sp., mainly on the females. The occurrence of Blastocystis sp. was 6% in symptomatic patients who required medical attendance in the time period between 2005 and 2013. Of the 80 patients, 73.75% indicated that they had gastrointestinal symptoms: 40 patients complained of abdominal pain and 17 with blood in their stool, while other symptoms, such as meteorism (15 subjects), weigh loss (8 subjects), perianal pain or itching (6 subjects), passing stool with mucus (5 subjects), vomiting (2 subjects) and fever (2 subjects) were less frequent. The prevalence of abdominal pain in the cohort without skin lesions was higher compared to those patients with skin problems (p = 0.007). The mean value of C-reactive protein showed elevated levels, but eosinophils were within a normal range. In addition, we did not find significant difference in eosinophilia between patients with vs. without skin manifestations. Thus, we suggest that eosinophilia is not an obligatory laboratory finding in protozoon infections, such as Blastocystis sp. In the light of our results, we suggest a stool parasite examination for patients with skin lesions of unknown origin.

Isolated sinusitis sphenoidalis caused by Trichoderma longibrachiatum in an immunocompetent patient with headache.

We present a case of isolated sinusitis sphenoidalis caused by Trichoderma longibrachiatum, an emerging causal agent of fungal infections with an often fatal outcome. A Trichoderma strain was isolated from secretion obtained from the sinus sphenoidalis of a rhinosinusitis patient and identified by sequence analysis of two loci as Trichoderma longibrachiatum from the Longibrachiatum Clade of the genus Trichoderma. T. longibrachiatum can trigger a fatal pathomechanism in immunodeficient patients, but only rarely causes disease in healthy people. The case presented is unique because the patient was not immunocompromised.

Evaluation of fungaemia infections in a Hungarian university hospital between 1996 and 2009.

The incidence of Candida species causing bloodstream infections in the University Hospital of Szeged, Hungary, between 1996 and 2009, and the susceptibilities of these isolates to antifungal agents were evaluated.Automated blood culture systems (Vital, bioMérieux, Marcy-l'Etoile, France; and BACTEC 9120, Becton-Dickinson Diagnostic Systems, Sparks, USA) were used. The in vitro susceptibilities of the yeast isolates to antifungal agents were determined by the Etest method (AB Biodisk, Solna, Sweden).Bloodstream infections were caused by yeast strains in 231 cases during this period, and 226 Candida strains were cultured from 216 candidaemia patients. Bloodstream infections caused by multiple Candida spp. were diagnosed almost every year. Of the 216 patients, 67 were children; and 55 infants needed intensive care. In 2005, C. glabrata caused an increase in the incidence of invasive fungal infections in the Neonatal Intensive Care Unit. The PFGE analysis of 12 isolates distinguished 4 different karyotypes. The incidence of bloodstream infections caused by fungi did not change during the 14-year study period. The most frequent species cultured from blood samples were C. albicans and C. glabrata. The incidence of resistant isolates remained constant. The local trends of fungaemia must be monitored and compared with global reports.

Molecular identification and antifungal susceptibilities of black Aspergillus isolates from otomycosis cases in Hungary.

Otomycosis, also known as fungal otitis externa, has been used to describe a fungal infection of the external auditory canal, but sometimes involving the middle ear. Many fungal species have been identified as infectious agents in otomycosis, with Aspergillus and Candida species being the most common. Among aspergilli, Aspergillus niger is the most commonly described species in the literature. In this study, 14 black Aspergillus strains were analyzed, which were isolated from otomycosis cases in Hungary between 2010 and 2011. These strains were identified as A. niger according to conventional morphological methods. Species identification was based on sequencing of part of the calmodulin gene. Our results indicate that instead of A. niger, A. awamori and A. tubingensis are the predominant species that cause ear infections in Southern Hungary. Antifungal susceptibility tests were carried out against four antifungal drugs: amphotericin B, itraconazole, ketoconazole and terbinafine. All isolates were found to exhibit low in vitro MIC values to amphotericin B, terbinafine and itraconazole. However, the examined isolates exhibited high in vitro MIC values to ketoconazole.

Keratitis caused by the recently described new species Aspergillus brasiliensis: two case reports.

INTRODUCTION: Human infections caused by Aspergillus brasiliensis have not yet been reported. We describe the first two known cases of fungal keratitis caused by Aspergillus brasiliensis. CASE PRESENTATIONS: A 49-year-old Indian Tamil woman agricultural worker came with pain and defective vision in the right eye for one month. Meanwhile, a 35-year-old Indian Tamil woman presented with a history of a corneal ulcer involving the left eye for 15 days. The fungal strains isolated from these two cases were originally suspected to belong to Aspergillus section Nigri based on macro- and micromorphological characteristics. Molecular identification revealed that both isolates represent A. brasiliensis. CONCLUSION: The two A. brasiliensis strains examined in this study were part of six keratitis isolates from Aspergillus section Nigri, suggesting that this recently described species may be responsible for a significant proportion of corneal infections caused by black Aspergilli. The presented cases also indicate that significant differences may occur between the severities of keratitis caused by individual isolates of A. brasiliensis.

Mycotic keratitis due to Aspergillus nomius.

We report the first known case of fungal keratitis caused by Aspergillus nomius. Ocular injury was known as a predisposing factor. The patient was treated with natamycin and econazole eye drops, itraconazole eye ointment, and oral ketoconazole. A therapeutic penetrating keratoplasty was performed 16 days after presentation. A sequence-based approach was used to assign the isolate to a species.

Infectious keratitis caused by Aspergillus tubingensis.

PURPOSE: To report 2 cases of keratomycosis caused by Aspergillus tubingensis. METHODS: The therapeutic courses were recorded for 2 male patients, 52 and 78 years old, with fungal keratitis caused by black Aspergillus strains. Morphological examination of the isolates was carried out on malt extract agar plates. A segment of the beta-tubulin gene was used for molecular identification. Antifungal susceptibilities were determined by the E test method for molds and the broth microdilution technique National Committee for Clinical Laboratory Standards M38-A. RESULTS: A 52-year-old man presented with complaints of pain and redness in the right eye. The patient was successfully treated with natamycin and econazole eyedrops, itraconazole eye ointment, and oral ketoconazole. A 78-year-old man presented with total corneal necrosis in the right eye. A therapeutic keratoplasty was performed, and topical natamycin and econazole were applied. At the postoperative visit after 3 weeks, almost the full corneal graft was clear with formed anterior chamber. Black Aspergillus strains were isolated from the corneal scrapings of both cases and initially identified as Aspergillus niger based on culture characteristics. Sequence analysis of a segment of the beta-tubulin gene revealed that the isolates are representatives of A. tubingensis. CONCLUSIONS: Aspergillus tubingensis is closely related with A. niger, the differentiation of these 2 species is difficult by classical morphological criteria. To our knowledge, the presented cases of fungal keratitis are the first reports on ocular infection caused by A. tubingensis.

Case of keratitis caused by Aspergillus tamarii.

We report a case of Aspergillus tamarii keratitis. Ocular injury was known to be a predisposing factor. Topical natamycin and econazole treatment and subsequent systemic ketoconazole treatment proved effective. The isolate was identified by morphological characteristics and sequence analysis as A. tamarii, a member of Aspergillus section Flavi not hitherto reported from keratomycosis.

Rapid discrimination between Candida albicans and Candida dubliniensis by using real-time polymerase chain reaction.

Several phenotypic methods have been used for the differentiation of Candida albicans and Candida dubliniensis, but molecular investigations are considered most reliable in their diagnostic value. Here, we suggest a rapid real-time polymerase chain reaction assay where the discrimination was achieved through melting point analysis with the help of the nonspecific fluorescent dye SybrGreen.

Occurrence and genetic variability of Candida parapsilosis sensu lato in Hungary.

The occurrence and genetic variability of Candida parapsilosis isolates in two Hungarian hospitals, located in Debrecen and Pécs, were examined. Among the 209 Candida isolates examined, 20 were found to belong to C. parapsilosis sensu lato, based on morphological, physiological and molecular data. The frequency of occurrence of C. parapsilosis isolates (9.6%) was lower than that observed in Europe but higher than that observed previously in Hungary. The genetic variability of C. parapsilosis sensu lato isolates was also examined using random amplified polymorphic DNA (RAPD) analysis and sequence analysis of the intergenic transcribed spacer (ITS) region of the rRNA gene cluster. The genetic variability of the isolates was relatively high, as revealed by RAPD analysis. Two isolates were found to belong to the recently described Candida metapsilosis species (C. parapsilosis group III), based on ITS sequence data, RAPD analysis and phenotypic data. These two isolates could also be distinguished from C. parapsilosis sensu stricto isolates using a primer pair developed for the detection of C. parapsilosis group I isolates. To the best of the authors' knowledge, this is the first report on the identification of C. metapsilosis from bloodstream infection.

Use of the BIOMIC Video System to evaluate the susceptibility of clinical yeast isolates to fluconazole and voriconazole by a disk diffusion method.

The ARTEMIS Global Antifungal Susceptibility Program provides the collection of epidemiological data and the results of the fluconazole and voriconazole susceptibility testing of yeast isolates. Participating in this study, a total of 7318 clinical yeast isolates were tested from different geographical areas in Hungary in the period 2001 to 2003. The species isolated most frequently was C. albicans (68.8%), followed by C. glabrata (11.8%), C. tropicalis (5.7%) and C. krusei (4.6%). Isolates of C. albicans, C. kefyr, C. lusitaniae, C. tropicalis and C. parapsilosis were highly susceptible to fluconazole (78.9-100%). The rates of isolation of fluconazole-resistant C. glabrata and C. krusei were higher in our study than the global mean in 2001 (28.2% and 87.5% vs. 18.3% and 70.2%, respectively). Differences were detected in the distribution of fluconazole-susceptibility data of C. glabrata isolates in the different counties of Hungary: most of the resistant isolates were observed in the eastern part of the country.

Comparative study of potential virulence factors in human pathogenic and saprophytic Trichoderma longibrachiatum strains.

Potential virulence factors of 9 saprophytic and 12 clinical Trichoderma longibrachiatum strains were examined in the present study, in order to compare their capacity to cause infection in humans. All of the strains were able to grow at temperatures up to 40 degrees C and at pH values ranging from 2.0 to 9.0. Carbon and nitrogen source utilization experiments revealed that all of the strains were able to utilize a series of basic amino acids both as sole carbon and nitrogen sources. The MIC values of the tested antifungal drugs were found to be 0.016-8 microg/ml for amphotericin B, 64-256 microg/ml for fluconazole, 0.5-32 microg/ml for itraconazole and 0.008-1 microg/ml for ketoconazole in the case of the examined isolates. Metabolites of the strains inhibited the growth of different bacteria, furthermore, compounds produced by three clinical isolates reduced the motility of boar spermatozoa, indicating their toxicity to mammalian cells as well. On the whole, there were no significant differences in the examined features between strains derived from clinical or soil samples. The question, however, whether all environmental Trichoderma longibrachiatum strains have the capacity to cause infections or not, remains still unanswered.

Etest for assessing the susceptibility of filamentous fungi.

The purpose of this study was to evaluate the Etest as an in vitro antifungal susceptibility test method for different moulds originating from human samples and from the environment. A total of 50 isolates (1 Acremonium, 18 Aspergillus, 2 Cladosporium, 1 Epicoccum, 15 Penicillium, 2 Scopulariopsis and 11 Trichoderma strains) were tested by the Etest. Forty-six of the tested moulds (92%) were resistant to fluconazole with minimal inhibitory concentrations (MICs) > or = 256 microg ml(-1). There were strains resistant to ketoconazole among Aspergillus niger, A. ochraceus and Cladosporium spp. with MICs > 32 microg ml(-1). For fluconazole, no differences were observed using two different inocula, while for itraconazole, ketoconazole and amphotericin B, a 1 or less step 2-fold dilution difference in MIC was seen for the most of 10 selected strains. The MICs of fluconazole and amphotericin B obtained for Trichoderma strains by the Etest and the agar dilution method were also compared. MICs for fluconazole were in agreement, while MICs for amphotericin B were higher with 1 or 2 steps of 2-fold dilutions for most of Trichoderma strains in the case of the agar dilution method.

Production of extracellular proteases by human pathogenic Trichoderma longibrachiatum strains.

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, and N-Succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens.