Fungi

JoF, Vol. 5, Pages 105: Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens.

JoF, Vol. 5, Pages 104: Shift in Epidemiology of Cryptococcal Infections in Ottawa with High Mortality in Non-HIV Immunocompromised Patients Cryptococcus neoformans is a fungus that can cause life-threatening infections. While human immunodeficiency virus (HIV)-positive status historically had the highest risk for cryptococcal infection and was associated with high mortality rates, there have been changes in HIV treatment and the epidemiology of other acquired immunodeficiencies, such as hematological malignancies. We conducted a retrospective case series analysis of patients who had cryptococcal infections documented at the Ottawa Hospital from 2005 to 2017. The Ottawa Hospital is a tertiary care hospital and provides complex care such as chemotherapy and transplantations. There were 28 confirmed cryptococcal infections. The most common underlying condition associated with cryptococcal infection was hematological malignancy (n = 8, 29%), followed by HIV (n = 5, 18%) and solid organ transplantation (n = 4, 14%). Furthermore, while there was a decrease in the number of cryptococcal infections in HIV patients after 2010 (four to one case), the number of cases in non-HIV immunocompromised patients increased from four in the years 2005–2010 to fourteen in 2011–2017. There were nine cryptococcal-attributable deaths. The case fatality rate was highest among patients with underlying hematological malignancies (63%), followed by solid organ transplant (50%) and HIV patients (20%). In conclusion, this study showed that there may be an epidemiological shift of cryptococcal infection in Ottawa. Additionally, infections may be associated with a worse prognosis in patients with a hematological malignancy and solid organ transplant than in patients with HIV infection in the modern era. Better prevention and/or treatment is warranted for high-risk populations.

JoF, Vol. 5, Pages 103: Thermogenic Characterization and Antifungal Susceptibility of Candida auris by Microcalorimetry Candida auris has emerged globally as a multidrug-resistant fungal pathogen. Isolates of C. auris are reported to be misidentified as Candida haemulonii. The aim of the study was to compare the heat production profiles of C. auris strains and other Candida spp. and evaluate their antifungal susceptibility using isothermal microcalorimetry. The minimum heat inhibitory concentrations (MHIC) and the minimum biofilm fungicidal concentration (MBFC) were defined as the lowest antimicrobial concentration leading to the lack of heat flow production after 24 h for planktonic cells and 48 h for biofilm-embedded cells. C. auris exhibited a peculiar heat production profile. Thermogenic parameters of C. auris suggested a slower growth rate compared to Candida lusitaniae and a different distinct heat profile compared to that of C. haemulonii species complex strains, although they all belong to the Metschnikowiaceae clade. Amphotericin B MHIC and MBFC were 0.5 µg/mL and ≥8 µg/mL, respectively. C. auris strains were non-susceptible to fluconazole at tested concentrations (MHIC > 128 µg/mL, MBFC > 256 µg/mL). The heat curve represents a fingerprint of C. auris, which distinguished it from other species. Treatment based on amphotericin B represents a potential therapeutic option for C. auris infection.

JoF, Vol. 5, Pages 102: Genetic Regulators and Physiological Significance of Glycogen Storage in Candida albicans The dimorphic human fungal pathogen C. albicans has broad metabolic flexibility that allows it to adapt to the nutrient conditions in different host habitats. C. albicans builds large carbohydrate stores (glycogen) at the end of exponential growth and begins consumption of stored carbohydrates when nutrients become limiting. The expression of genes required for the successful transition between host environments, including the factors controlling glycogen content, is controlled by protein kinase A signaling through the transcription factor Efg1. In addition to the inability to transition to hyphal growth, C. albicans efg1 mutants have low glycogen content and reduced long-term survival, suggesting that carbohydrate storage is required for viability during prolonged culture. To test this assumption, we constructed a glycogen-deficient C. albicans mutant and assessed its viability during extended culture. Pathways and additional genetic factors controlling C. albicans glycogen synthesis were identified through the screening of mutant libraries for strains with low glycogen content. Finally, a part of the Efg1-regulon was screened for mutants with a shortened long-term survival phenotype. We found that glycogen deficiency does not affect long-term survival, growth, metabolic flexibility or morphology of C. albicans. We conclude that glycogen is not an important contributor to C. albicans fitness.

JoF, Vol. 5, Pages 101: Ongoing Challenges with Healthcare-Associated Candida auris Outbreaks in Oman Candida auris has emerged in the past decade as a multi-drug resistant public health threat causing health care outbreaks. Here we report epidemiological, clinical, and microbiological investigations of a C. auris outbreak in a regional Omani hospital between April 2018 and April 2019. The outbreak started in the intensive care areas (intensive care unit (ICU), coronary care unit (CCU), and high dependency unit) but cases were subsequently diagnosed in other medical and surgical units. In addition to the patients’ clinical and screening samples, environmental swabs from high touch areas and from the hands of 35 staff were collected. All the positive samples from patients and environmental screening were confirmed using MALDI-TOF, and additional ITS-rDNA sequencing was done for ten clinical and two environmental isolates. There were 32 patients positive for C. auris of which 14 (43.8%) had urinary tract infection, 11 (34.4%) had candidemia, and 7 (21.8%) had asymptomatic skin colonization. The median age was 64 years (14–88) with 17 (53.1%) male and 15 (46.9%) female patients. Prior to diagnosis, 21 (65.6%) had been admitted to the intensive care unit, and 11 (34.4%) had been nursed in medical or surgical wards. The crude mortality rate in our patient’s cohort was 53.1. Two swabs collected from a ventilator in two different beds in the ICU were positive for C. auris. None of the health care worker samples were positive. Molecular typing showed that clinical and environmental isolates were genetically similar and all belonged to the South Asian C. auris clade I. Most isolates had non-susceptible fluconazole (100%) and amphotericin B (33%) minimal inhibitory concentrations (MICs), but had low echinocandin and voriconazole MICs. Despite multimodal infection prevention and control measures, new cases continued to appear, challenging all the containment efforts.

JoF, Vol. 5, Pages 100: PdMFS1 Transporter Contributes to Penicilliun digitatum Fungicide Resistance and Fungal Virulence during Citrus Fruit Infection A new Penicillium digitatum major facilitator superfamily (MFS) transporter (PdMFS1) was identified and functionally characterized in order to shed more light on the mechanisms underlying fungicide resistance. PdMFS1 can play an important role in the intensification of resistance to fungicides normally used in P. digitatum postharvest treatments. In the PdMFS1 disrupted mutants, a slight effect in response to chemical fungicides was observed, but fungicide sensitivity was highly affected in the overexpression mutants which became resistant to wide range of chemical fungicides. Moreover, P. digitatum knock-out mutants exhibited a lower rate of fungal virulence when infected oranges were stored at 20 °C. Disease symptoms were higher in the PdMFS1 overexpression mutants coming from the low-virulent P. digitatum parental strain. In addition, the gene expression analysis showed an induction of PdMFS1 transcription in all overexpression mutants regardless from which progenitor came from, and four-time intensification of the parental wild type strain during citrus infection reinforcing PdMFS1 role in fungal virulence. The P. digitatum MFS transporter PdMFS1 contributes not only to the acquisition of wide range of fungicide resistance but also in fungal virulence during citrus infection.

JoF, Vol. 5, Pages 99: 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.

JoF, Vol. 5, Pages 98: Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes Mucormycosis infections are infrequent yet aggressive and serious fungal infections. Early diagnosis of mucormycosis and its discrimination from other fungal infections is required for targeted treatment and more favorable patient outcomes. The majority of the molecular assays use 18 S rDNA. In the current study, we aimed to explore the potential of the mitochondrial rnl (encoding for large-subunit-ribosomal-RNA) gene as a novel molecular marker suitable for research and diagnostics. Rnl was evaluated as a marker for: (1) the Mucorales family, (2) species identification (Rhizopus arrhizus, R. microsporus, Mucor circinelloides, and Lichtheimia species complexes), (3) growth stage, and (4) quantification. Sensitivity, specificity, discriminatory power, the limit of detection (LoD), and cross-reactivity were evaluated. Assays were tested using pure cultures, spiked clinical samples, murine organs, and human paraffin-embedded-tissue (FFPE) samples. Mitochondrial markers were found to be superior to nuclear markers for degraded samples. Rnl outperformed the UMD universal® (Molyzm) marker in FFPE (71.5% positive samples versus 50%). Spiked blood samples highlighted the potential of rnl as a pan-Mucorales screening test. Fungal burden was reproducibly quantified in murine organs using standard curves. Identification of pure cultures gave a perfect (100%) correlation with the detected internal transcribed spacer (ITS) sequence. In conclusion, mitochondrial genes, such as rnl, provide an alternative to the nuclear 18 S rDNA genes and deserve further evaluation.

JoF, Vol. 5, Pages 97: Antifungal Drugs: Special Problems Treating Central Nervous System Infections Treating fungal infections in the central nervous system (CNS) remains a challenge despite the availability of new antifungal agents. Therapy is limited by poor understanding of the kinetic properties of antifungal drugs in the CNS compounded by lack of data for many agents. In some cases, clinical response rates do not correspond to data on drug concentrations in the cerebral spinal fluid and/or brain parenchyma. In order to better characterize the use of antifungal agents in treating CNS infections, a review of the essential principles of neuroPK are reviewed. Specific data regarding antifungal drug concentrations in the cerebral spinal fluid and brain tissue are described from human data where available. Alternative dosing regimens and the role of antifungal drug concentration monitoring in treating fungal infections in the CNS are also discussed. Having a better understanding of these key concepts will help guide clinicians in determining the best treatment courses for patients with these devastating infections.

JoF, Vol. 5, Pages 96: Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum Penicillium brevicompactum is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to P. brevicompactum. Although a generally available phleomycin resistance marker (ble) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette (nat1). To circumvent this failure, we constructed a new nat gene, considering the codon bias for P. brevicompactum. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, MAT1-2-1 and flbA. For MAT1-2-1, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used ble to reintroduce the wild-type genes. This step restored the wild-type phenotype in the flbA deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus P. brevicompactum.