Showing papers in "Medical Mycology in 2018"

Challenges in the diagnosis and treatment of mucormycosis.

Abstract: The diagnosis and treatment of mucormycosis are challenging. The incidence of the disease seems to be increasing. Hematological malignancies are the most common underlying disease in countries with high income and uncontrolled diabetes in developing countries. Clinical approach to diagnosis lacks sensitivity and specificity. Radiologically, multiple (≥10) nodules and pleural effusion are reportedly associated with pulmonary mucormycosis. Another finding on computerized tomography (CT) scan, which seems to indicate the presence of mucormycosis, is the reverse halo sign. Microscopy (direct and on histopathology) and culture are the cornerstones of diagnosis. Molecular assays can be used either for detection or identification of mucormycetes, and they can be recommended as valuable add-on tools that complement conventional diagnostic procedures. Successful management of mucormycosis is based on a multimodal approach, including reversal or discontinuation of underlying predisposing factors, early administration of active antifungal agents at optimal doses, complete removal of all infected tissues, and use of various adjunctive therapies. Our armamentarium of antifungals is slightly enriched by the addition of two newer azoles (posaconazole and isavuconazole) to liposomal amphotericin B, which remains the drug of choice for the initial antifungal treatment, according to the recently published guidelines by ECIL-6, as well as those published by ECMM/ESCMID. Despite the efforts for better understanding of the pathogenesis, early diagnosis and aggressive treatment of mucormycosis, the mortality rate of the disease remains high.

Malassezia ecology, pathophysiology, and treatment.

Abstract: Malassezia are lipid dependent basidiomycetous yeasts that inhabit the skin and mucosa of humans and other warm-blooded animals, and are a major component of the skin microbiome. They occur as skin commensals, but are also associated with various skin disorders and bloodstream infections. The genus currently comprises 17 species and has recently been assigned its own class, Malasseziomycetes. Importantly, multiple Malassezia species and/or genotypes may cause unique or similar pathologies and vary in their antifungal susceptibility. In addition to culture-based approaches, culture-independent methods have added to our understanding of Malassezia presence and abundance and their relationship to pathogenicity. Moreover, these novel approaches have suggested a much wider-spread presence, including other human body parts and even other ecosystems, but their role in these arenas requires further clarification. With recent successful transformation and genetic engineering of Malassezia, the role of specific genes in pathogenesis can now be studied. We suggest that characterizing the metabolic impact of Malassezia communities rather than species identification is key in elucidation of pathophysiological associations. Finally, the increasing availability of genome sequences may provide key information aiding faster diagnostics, and understanding of the biochemical mechanisms for Malassezia skin adaptation and the design of future drugs.

Scedosporium and Lomentospora: an updated overview of underrated opportunists.

Abstract: Species of Scedosporium and Lomentospora are considered as emerging opportunists, affecting immunosuppressed and otherwise debilitated patients, although classically they are known from causing trauma-associated infections in healthy individuals. Clinical manifestations range from local infection to pulmonary colonization and severe invasive disease, in which mortality rates may be over 80%. These unacceptably high rates are due to the clinical status of patients, diagnostic difficulties, and to intrinsic antifungal resistance of these fungi. In consequence, several consortia have been founded to increase research efforts on these orphan fungi. The current review presents recent findings and summarizes the most relevant points, including the Scedosporium/Lomentospora taxonomy, environmental distribution, epidemiology, pathology, virulence factors, immunology, diagnostic methods, and therapeutic strategies.

Diabetes mellitus as the major risk factor for mucormycosis in Mexico: Epidemiology, diagnosis, and outcomes of reported cases.

Abstract: Mucormycosis is an emerging infectious disease with high rates of associated mortality and morbidity. Little is known about the characteristics of mucormycosis or entomophthoromycosis occurring in Mexico. A search strategy was performed of literature published in journals found in available databases and theses published online at Universidad Nacional Autonoma de Mexico (UNAM) library website reporting clinical cases or clinical case series of mucormycosis and entomophthoromycosis occurring in Mexico between 1982 and 2016. Among the 418 cases identified, 72% were diabetic patients, and sinusitis accounted for 75% of the reported cases. Diabetes mellitus was not a risk factor for entomophthoromycosis. Mortality rate was 51% (125/244). Rhizopus species were the most frequent isolates (59%, 148/250). Amphotericin B deoxycholate was used in 89% of cases (204/227), while surgery and antifungal management as combined treatment was used in 90% (172/191). In diabetic individuals, this combined treatment approach was associated with a higher probability of survival (95% vs 66%, OR = 0.1, 95% CI, 0.02-0.43' P = .002). The most common complications were associated with nephrotoxicity and prolonged hospitalization due to IV antifungal therapy. An algorithm is proposed to establish an early diagnosis of rhino-orbital cerebral (ROC) mucormycosis based on standardized identification of warning signs and symptoms and performing an early direct microbiological exam and histopathological identification through a multidisciplinary medical and surgical team. In summary, diabetes mellitus was the most common risk factor for mucormycosis in Mexico; combined antifungal therapy and surgery in ROC mucormycosis significantly improved survival.

Fungal infections in animals: A patchwork of different situations

Abstract: The importance of fungal infections in both human and animals has increased over the last decades. This article represents an overview of the different categories of fungal infections that can be encountered in animals originating from environmental sources without transmission to humans. In addition, the endemic infections with indirect transmission from the environment, the zoophilic fungal pathogens with near-direct transmission, the zoonotic fungi that can be directly transmitted from animals to humans, mycotoxicoses and antifungal resistance in animals will also be discussed. Opportunistic mycoses are responsible for a wide range of diseases from localized infections to fatal disseminated diseases, such as aspergillosis, mucormycosis, candidiasis, cryptococcosis and infections caused by melanized fungi. The amphibian fungal disease chytridiomycosis and the Bat White-nose syndrome are due to obligatory fungal pathogens. Zoonotic agents are naturally transmitted from vertebrate animals to humans and vice versa. The list of zoonotic fungal agents is limited but some species, like Microsporum canis and Sporothrix brasiliensis from cats, have a strong public health impact. Mycotoxins are defined as the chemicals of fungal origin being toxic for warm-blooded vertebrates. Intoxications by aflatoxins and ochratoxins represent a threat for both human and animal health. Resistance to antifungals can occur in different animal species that receive these drugs, although the true epidemiology of resistance in animals is unknown, and options to treat infections caused by resistant infections are limited.

Sporotrichosis between 1898 and 2017: The evolution of knowledge on a changeable disease and on emerging etiological agents.

Abstract: The description of cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. Sporothrix schenckii causes a benign chronic subcutaneous mycosis, Sporothrix brasiliensis is highly virulent, and Sporothrix globosa mainly causes fixed cutaneous lesions. Furthermore, S. brasiliensis is the prevalent species related to cat-transmitted sporotrichosis. Sources of infection, transmission, and distribution patterns also differ between species, and variability differs between species because of different degrees of clonality. The present review article will cover several aspects of the biology of clinically relevant agents of sporotrichosis, including epidemiological aspects of emerging species. Genomic information of Sporothrix spp. is also discussed. The cell wall is an essential structure for cell viability, interaction with the environment, and the host immune cells and contains several macromolecules involved in virulence. Due to its importance, aspects of glycosylation and cell wall polysaccharides are reviewed. Recent genome data and bioinformatics analyses helped to identify specific enzymes of the biosynthetic glycosylation routes, with no homologs in mammalian cells, which can be putative targets for development of antifungal drugs. A diversity of molecular techniques is available for the recognition of the clinically relevant species of Sporothrix. Furthermore, antigens identified as diagnostic markers and putative vaccine candidates are described. Cell-mediated immunity plays a key role in controlling infection, but Sporothrix species differ in their interaction with the host. The adaptive branch of the immune response is essential for appropriate control of infection.

Triazole resistance surveillance in Aspergillus fumigatus

Abstract: Triazole resistance is an increasing concern in the opportunistic mold Aspergillus fumigatus. Resistance can develop through exposure to azole compounds during azole therapy or in the environment. Resistance mutations are commonly found in the Cyp51A-gene, although other known and unknown resistance mechanisms may be present. Surveillance studies show triazole resistance in six continents, although the presence of resistance remains unknown in many countries. In most countries, resistance mutations associated with the environment dominate, but it remains unclear if these resistance traits predominately migrate or arise locally. Patients with triazole-resistant aspergillus disease may fail to antifungal therapy, but only a limited number of cohort studies have been performed that show conflicting results. Treatment failure might be due to diagnostic delay or due to the limited number of alternative treatment options. The ISHAM/ECMM Aspergillus Resistance Surveillance working group was set up to facilitate surveillance studies and stimulate international collaborations. Important aims are to determine the resistance epidemiology in countries where this information is currently lacking, to gain more insight in the clinical implications of triazole resistance through a registry and to unify nomenclature through consensus definitions.

Survey of laboratory practices for diagnosis of fungal infection in seven Asian countries: An Asia Fungal Working Group (AFWG) initiative.

Abstract: An online survey of mycology laboratories in seven Asian countries was conducted to assess the status, competence, and services available. Country representatives from the Asia Fungal Working Group (AFWG) contacted as many laboratories performing mycology diagnosis as possible in their respective countries, requesting that the laboratory heads complete the online survey. In total, 241 laboratories responded, including 71 in China, 104 in India, 11 in Indonesia, 26 in the Philippines, four in Singapore, 18 in Taiwan, and seven in Thailand. Overall, 129/241 (53.5%) surveyed mycology laboratories operate as separate designated mycology laboratories, 75/241 (31.1%) conduct regular formal staff training, 103/241 (42.7%) are accredited, and 88/157 (56.1%) participate in external quality assurance scheme (EQAS) programs. Microscopy and culture methods are available in nearly all laboratories, although few perform DNA sequencing (37/219; 16.9%) or use matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) (27/219; 12.3%) for isolate identification. Antifungal susceptibility testing is performed in 142/241 (58.9%) laboratories, mainly for yeasts. The most commonly performed nonculture diagnostic is cryptococcal antigen testing (66 laboratories), followed by galactomannan testing (55), polymerase chain reaction (PCR) diagnosis (37), and beta-D-glucan testing (24). Therapeutic drug monitoring is conducted in 21 laboratories. There is almost no access to advanced diagnostic tests, like galactomannan, β-D-glucan, and PCR, in the surveyed laboratories in Indonesia, the Philippines, and Thailand. These results highlight the need for development of quality laboratories, accreditation and training of manpower in existing laboratories, and access to advanced non-culture-based diagnostic tests to facilitate the diagnosis of fungal infections in Asia.

Oral fungal-bacterial biofilm models in vitro: a review.

Abstract: Inclusion of fungi as commensals in oral biofilm is an important innovation in oral biology, and this work aimed to review the literature on the available biofilm and related disease in vitro models. Actually, thousands of bacterial and around one hundred of fungal phylotypes can colonize the oral cavity. Taxonomic profiling combined with functional expression analysis has revealed that Candida albicans, Streptococcus mutans and prominent periodontopathogens are not always present or numerically important in candidiasis, caries, or periodontitis lesions. However, C. albicans combined with Streptococcus spp. co-increase their virulence in invasive candidiasis, early childhood caries or peri-implantitis. As Candida species and many other fungi are also members of oral microcosms in healthy individuals, mixed fungal-bacterial biofilm models are increasingly valuable investigative tools, and new fungal-bacterial species combinations need to be investigated. Here we review the key points and current methods for culturing in vitro mixed fungal-bacterial models of oral biofilms. According to ecosystem under study (health, candidiasis, caries, periodontitis), protocol design will select microbial strains, biofilm support (polystyrene plate, cell culture, denture, tooth, implant), pre-treatment support (human or artificial saliva) and culture conditions. Growing mixed fungal-bacterial biofilm models in vitro is a difficult challenge. But reproducible models are needed, because oral hygiene products, food and beverage, medication, licit and illicit drugs can influence oral ecosystems. So, even though most oral fungi and bacteria are not cultivable, in vitro microbiological models should still be instrumental in adapting oral care products, dietary products and care protocols to patients at higher risk of oral diseases. Microbial biofilm models combined with oral epithelial cell cultures could also aid in understanding the inflammatory reaction.

Dermatophytes and dermatophytosis in the eastern and southern parts of Africa.

Abstract: Dermatophytosis is currently a disease of global importance and a public health burden. It is caused by dermatophytes, which attack and grow on dead animal keratin. Dermatophytes belong to three genera, namely, Epidermophyton, Microsporum, and Trichophyton. The predominant clinical forms and causative agents vary from one region of the world to another. Poor socioeconomic status, high population densities, and poor sanitary conditions are some of the factors responsible for the high prevalence of dermatophytosis in many developing countries, which include countries in southern and eastern Africa, the focus of this review. To the best of our knowledge, there is currently no review article on published findings on dermatophytosis in the eastern and southern parts of Africa. This information will be of interest to the medical and research community since the world has become a global village. This review covers published research findings in eastern and southern regions of Africa until this date. The countries covered in the current review include Kenya, Ethiopia, Tanzania, South Africa, Mozambique, Madagascar, Malawi, Rwanda, Burundi, Uganda, Zambia, Zimbabwe, and Botswana. T. violaceum is the most common human etiological agent in all the countries under review with prevalence ranging from 56.7% to 95%, except for Madagascar (M. langeronii, reclassified as M. audouinii), Uganda (M. gypseum) and Malawi (M. audouinii). Tinea capitis was the most clinical type, followed by tinea corporis. Etiological agents of animal dermatophytoses were variable in the countries where they were reported. Major risk factors for dermatophytoses are age, climatic, and socioeconomic factors.

Closing the mycetoma knowledge gap.

Abstract: On 28th May 2016, mycetoma was recognized as a neglected tropical disease by the World Health Organization. This was the result of a 4-year journey starting in February 2013 with a meeting of global mycetoma experts. Knowledge gaps were identified and included the incidence, prevalence, and mapping of mycetoma; the mode of transmission; the development of methods for early diagnosis; and better treatment. In this review, we review the road to recognition, the ISHAM working group meeting in Argentina, and we address the progress made in closing the knowledge gaps since 2013. Progress included adding another 9000 patients to the literature, which allowed us to update the prevalence map on mycetoma. Furthermore, based on molecular phylogeny, species names were corrected and four novel mycetoma causative agents were identified. By mapping mycetoma causative agents an association with Acacia trees was found. For early diagnosis, three different isothermal amplification techniques were developed, and novel antigens were discovered. To develop better treatment strategies for mycetoma patients, in vitro susceptibility tests for the coelomycete agents of black grain mycetoma were developed, and the first randomized clinical trial for eumycetoma started early 2017.

Epidemiology of human dermatophytoses in Africa.

Abstract: In this critical literature review, we summarize the epidemiological trends of dermatophytoses reported in Africa. Our findings clearly emphasize the heavy burden of dermatophytosis in Africa. Tinea capitis is the primary clinical presentation of dermatophytosis in African children throughout the entire African continent. The disease affects more than 20% of school-age children in West Africa, while the prevalence ranges from 10% to more than 70% in other regions of Africa. In African adults, the presence of tinea corporis is the most frequent indicator of dermatophytosis. However, epidemiological studies have been primarily conducted on particular patient groups that are not representative of the general population. We examined dermatophyte species distribution patterns. We observed a predominance of anthropophilic dermatophytes, mainly T. violaceum, in the North and East of Africa and both T. soudanense and M. audouinii in the Western and Central regions of the continent. Interestingly, the zoophilic species, M. canis, has recently emerged in North and East Africa. Optimization of both mycology diagnosis capacities and epidemiological methodology would provide insight into the role that climate and other global aspects of the human environment play in dermatophyte epidemiology. We advocate that using a multisectoral and collaborative strategy would strengthen such future studies.

In vitro activity of the novel antifungal compound F901318 against Australian Scedosporium and Lomentospora fungi.

Abstract: We determined the in vitro activity of the novel orotomide antifungal, F901318, against 30 Lomentospora prolificans, 20 Scedosporium apiospermum, 7 S. aurantiacum, and 3 S. boydii, isolates in comparison with standard antifungals. Against L. prolificans, F901318 was the most potent compound (MIC90 0.25 μg/ml); the geometric mean MIC (0.26 μg/ml) was significantly lower (23-80-fold) than those of itraconazole, voriconazole, posaconazole, and isavuconazole (all P < .001), and amphotericin B (P < .05). F901318 also had good activity against S. apiospermum, S. aurantiacum, and S. boydii, comparable to that of voriconazole and posaconazole but was more active than isavuconazole for all three species.

New insights in dermatophyte research.

Abstract: Dermatophyte research has renewed interest because of changing human floras with changing socioeconomic conditions, and because of severe chronic infections in patients with congenital immune disorders. Main taxonomic traits at the generic level have changed considerably, and now fine-tuning at the species level with state-of-the-art technology has become urgent. Research on virulence factors focuses on secreted proteases now has support in genome data. It is speculated that most protease families are used for degrading hard keratin during nitrogen recycling in the environment, while others, such as Sub6 may have emerged as a result of ancestral gene duplication, and are likely to have specific roles during infection. Virulence may differ between mating partners of the same species and concepts of zoo- and anthropophily may require revision in some recently redefined species. Many of these questions benefit from international cooperation and exchange of materials. The aim of the ISHAM Working Group Dermatophytes aims to stimulate and coordinate international networking on these fungi.

Limonene inhibits Candida albicans growth by inducing apoptosis

Abstract: Anti-Candida potential of limonene was evaluated against planktonic growth, biofilm (adhesion, development and maturation) and morphogenesis of Candida albicans in this study. Limonene is a major constituent of citrus oil and most frequently used terpene in food and beverage industry due to its pleasant fragrance, nontoxic, and is generally recognized as safe (GRAS) flavoring agent as well as treatment option in many gastrointestinal diseases.Limonene exhibited excellent anti-Candida activity and was equally effective against planktonic growth of C. albicans isolates differentially susceptible to FLC (N = 35). Limonene inhibited morphogenesis significantly at low concentration. However, it showed stage dependent activity against biofilm formation, that is, it was more effective against adhesion followed by development and maturation. Limonene also exhibited excellent synergy with FLC against planktonic and biofilm growth. SWATH-MS analysis led to identification of limonene responsive proteins that provided molecular insight of its anti-Candida activity. Proteomic analysis revealed upregulation of proteins involved in cell wall glucan synthesis (Kre6); oxidative stress (Rhr2, Adh7 and Ebp1); DNA damage stress (Mbf1 and Npl3); nucleolar stress (Rpl11, Rpl7, Rpl29, Rpl15) and down regulation of cytoskeleton organization (Crn1, Pin3, Cct8, Rbl2), and so forth, in response to limonene. Limonene mediated down regulation of Tps3 indicates activation of caspase (CaMca1) and induction of apoptosis in C. albicans. These results suggest that limonene inhibits C. albicans growth by cell wall/membrane damage induced oxidative stress that leads to DNA damage resulting into modulation of cell cycle and induction of apoptosis through nucleolar stress and metacaspase dependent pathway.

Ecology of Paracoccidioides brasiliensis, P. lutzii and related species: infection in armadillos, soil occurrence and mycological aspects.

Abstract: Paracoccidioides brasiliensis and the related species P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii (Ascomycota, Ajellomycetaceae) are the etiological agents of paracoccidoidoimycosis (PCM), one of the most important systemic mycoses in Latin America. They are dimorphic fungi, with a mycelial life cycle in soil and a yeast phase associated with tissues of mammalian hosts. This study aimed to detect Paracoccidioides spp. in armadillo tissues and associated soil samples in three well-defined geographic areas, including the Alta Floresta, an area not only endemic for PCM in the central region of Brazil but also of probable P. lutzii occurrence, whose ecology and geographic distribution are poorly elucidated. The isolates were genotyped by sequencing ITS-rDNA and the gp43-exon-2 region, and by PCR-RFLP of alpha tubulin (tub1) gene; mycological aspects such as yeast-to-mycelial transition, growth and conidial production in soil extract agar were also evaluated. We confirmed that while armadillos are highly infected by P. brasiliensis, including multiple infections by distinct genotypes or species (P. brasiliensis and P. americana) in the same animal, the same does not hold true for P. lutzii, which in turn seems to present less capacity for mycelial growth and conidial production, when developing in a soil-related condition.

Aspergillus terreus: Novel lessons learned on amphotericin B resistance.

Abstract: The polyene antifungal amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and in general displays a remarkably low rate of antimicrobial resistance. Aspergillus terreus holds an exceptional position among the Aspergilli due to its intrinsic AmB resistance, in vivo and in vitro. Until now, the underlying mechanisms of polyene resistance were not well understood. This review will highlight the molecular basis of A. terreus and AmB resistance recently gained and will display novel data on the mode of action of AmB. A main focus is set on fundamental stress response pathways covering the heat shock proteins (Hsp) 90/Hsp70 axis, as well as reactive oxygen species detoxifying enzymes in response to AmB. The effect on main cellular functions such as fungal respiration will be addressed in detail and resistance mechanisms will be highlighted. Based on these novel findings we will discuss new molecular targets for alternative options in the treatment of invasive infections due to A. terreus.

Candida vaginitis: virulence, host response and vaccine prospects

Abstract: Vulvovaginal candidiasis is a common mucosal infection affecting a large proportion of women with some of them affected by recurrent often intractable forms of the disease. Thus, there is an increasing interest in understanding the pathogenesis of this disease. The aim of our work was to characterize, in animal models of vaginal candidiasis, the components of the host-fungus interaction at the mucosal level.The evidence of an immune response in the vaginal compartment was very encouraging to identify the proper targets for new strategies for vaccination or immunotherapy of vaginal candidiasis. Aspartyl-proteinase (Sap2), which is an important immunodominant antigens and virulence factors of C.albicans acting in mucosal infections, was assembled with virosomes and a vaccine PEV7 was obtained. The results obtained in the mouse model and in the clinical trial conducted by Pevion on women have evidenced that the vaccine PEV7, intravaginally administered, has an encouraging therapeutic potential for the treatment of recurrent vulvovaginal candidiasis. This opens the way to a modality for anti-Candida protection at mucosal level.

Epidemiology and clinical characteristics of invasive mould infections: A multicenter, retrospective analysis in five Asian countries.

Abstract: Formal, large-scale, multicenter studies of invasive mould infection (IMI) in Asia are rare This 1-year, retrospective study was designed to assess the incidence and clinical determinants of IMI in centers in five countries (Thailand, Taiwan, Singapore, China, India) Patients treated in a single year (2012) were identified through discharge diagnoses, microbiology, and histopathology logs, and entered based on published definitions of IMI A total of 155 cases were included (median age 54 years; 477% male) Of these, 477% had proven disease; the remainder had probable IMI The most frequent host factors were prolonged steroid use (394%) and recent neutropenia (387%) Common underlying conditions included diabetes mellitus (DM; 309%), acute myeloid leukemia (194%), and rheumatologic conditions (116%) DM was more common in patients with no recent history of neutropenia or prolonged steroid use (P = 006) The lung was the most frequently involved site (787%), demonstrating a range of features on computed tomography (CT) Aspergillus was the most common mould cultured (716%), primarily A fumigatus and A flavus, although proportions varied in different centers The most often used antifungal for empiric therapy was conventional amphotericin Ninety-day mortality was 329% This is the first multicenter Asian study of IMI not limited to specific patient groups or diagnostic methods It suggests that DM and rheumatologic conditions be considered as risk factors for IMI and demonstrates that IMI should not be ruled out in patients whose chest features on CT do not fit the conventional criteria

Diagnosis of aspergillosis by PCR: Clinical considerations and technical tips.

Abstract: Standardization of Aspergillus polymerase chain reaction (PCR) protocols has progressed, and analytical validity of blood-based assays has been formally established. It remains necessary to consider how the tests can be used in practice to maximize clinical utility. To determine the optimal diagnostic strategies and influence on patient management, several factors require consideration, including the patient population, incidence of invasive aspergillosis (and other fungal disease), and the local antifungal prescribing policy. Technical issues such as specimen type, ease of sampling, frequency of testing, access to testing centers, and time to reporting will also influence the use of PCR in clinical practice. Interpretation of all diagnostic tests is dependent on the clinical context and molecular assays are no exception, but with the proposal to incorporate Aspergillus PCR into the second revision of the consensus guidelines for defining invasive fungal disease the acceptance and understanding of molecular tests should improve.

Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Abstract: An expanded library of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been constructed using the spectra generated from 42 clinical isolates and 11 reference strains, including 23 different species from 8 sections (16 cryptic plus 7 noncryptic species). Out of a total of 379 strains of Aspergillus isolated from clinical samples, 179 strains were selected to be identified by sequencing of beta-tubulin or calmodulin genes. Protein spectra of 53 strains, cultured in liquid medium, were used to construct an in-house reference database in the MALDI-TOF MS. One hundred ninety strains (179 clinical isolates previously identified by sequencing and the 11 reference strains), cultured on solid medium, were blindy analyzed by the MALDI-TOF MS technology to validate the generated in-house reference database. A 100% correlation was obtained with both identification methods, gene sequencing and MALDI-TOF MS, and no discordant identification was obtained. The HUVR database provided species level (score of ≥2.0) identification in 165 isolates (86.84%) and for the remaining 25 (13.16%) a genus level identification (score between 1.7 and 2.0) was obtained. The routine MALDI-TOF MS analysis with the new database, was then challenged with 200 Aspergillus clinical isolates grown on solid medium in a prospective evaluation. A species identification was obtained in 191 strains (95.5%), and only nine strains (4.5%) could not be identified at the species level. Among the 200 strains, A. tubingensis was the only cryptic species identified. We demonstrated the feasibility and usefulness of the new HUVR database in MALDI-TOF MS by the use of a standardized procedure for the identification of Aspergillus clinical isolates, including cryptic species, grown either on solid or liquid media.

Molecular identification of Trichophyton benhamiae in Strasbourg, France: a 9-year retrospective study.

Abstract: Trichophyton benhamiae is a zoophilic dermatophyte transmitted to humans mostly from guinea pigs and occasionally other animals. It presents two distinct phenotypes: yellow and white. T. benhamiae was formerly known as Trichophyton species of Arthroderma benhamiae; it was considered part of the T. mentagrophytes species complex, and some authors have incorrectly described the yellow phenotype of T. benhamiae as T. mentagrophytes var. porcellae. Identification of T. benhamiae has been difficult, as it was described under more than three names, two phenotypes, and in several different possible host species. During the past 15 years, human infections due to this dermatophyte have been increasingly reported all over the world. In order to better understand the local epidemiology of T. benhamiae and to compare it to other European countries, we performed a 9-year retrospective study in the Strasbourg University Hospital. We studied 41 dermatophytes (38 isolated from humans and 3 from guinea pigs) identified as T. mentagrophytes var. porcellae or A. benhamiae from January 2008 to December 2016 and verified their identification by ITS (Internal Transcribed Spacer) sequencing. ITS sequencing was performed in 35 of the 41 strains, and they were identified as T. benhamiae (33), T. bullosum (1), and T. eriotrephon (1). The other six remaining strains were identified according to morphology as T. mentagrophytes var. porcellae, name incorrectly used since 2010 for the yellow phenotype of T. benhamiae. ITS sequencing is recommended for accurate identification of this dermatophyte and the culture phenotype (yellow or white) should be specified.

Medical phycology 2017.

Abstract: In 2014, ISHAM formed a new working group: "Medical Phycology: Protothecosis and Chlorellosis." The purpose of this working group is to help facilitate collaboration and communication among people interested in the pathogenic algae, to share ideas and work together. Here we present reports on recent work we have done in five areas. 1. The history of medical phycology as a branch of science. 2. Aspects of the genetics of Prototheca. 3. Aspects of the proteins of Prototheca. 4. Human infections caused by Prototheca. 5. Dairy cow mastitis caused by Prototheca.

Biofilm, adherence, and hydrophobicity as virulence factors in Malassezia furfur.

Abstract: Malassezia species are natural inhabitants of the healthy skin. However, under certain conditions, they may cause or exacerbate several skin diseases. The ability of this fungus to colonize or infect is determined by complex interactions between the fungal cell and its virulence factors. This study aims to evaluate "in vitro" the hydrophobicity levels, the adherence on a plastic surface and the biofilm formation of 16 clinical isolates of Malassezia furfur. Cellular surface hydrophobicity (CSH) levels were determined by two-phase system. The biofilm formation was determined by tetrazolium salt (XTT) reduction assay and by Scanning Electron Microscopy (SEM). Results showed many isolates were hydrophobic, adherent, and producers of biofilm on abiotic surfaces with different capacity. SEM observations confirmed an abundant extracellular matrix after 48 h of biofilm formation. About 63% of strains with high production of biofilm showed medium to high percentage of hydrophobicity and/or adherence. In addition, it has been demonstrated a correlation between hydrophobicity, adherence, and biofilm formation in about 60% of strains examined. These important virulence factors could be responsible of this yeast changing from a commensal to a pathogenic status.

Antifungal susceptibility of Sporothrix schenckii complex biofilms.

Abstract: Sporotrichosis, caused by species of Sporothrix schenckii complex, is the most prevalent subcutaneous mycosis in many areas of Latin America. The aim of this study was to evaluate the ability of Sporothrix spp. to form biofilms in vitro and to characterize the growth kinetics, morphology, and antifungal susceptibility of biofilms against classical antifungals. We investigated the ability of strains to produce biofilms in vitro and determined the effects of exposure to amphotericin B, itraconazole, caspofungin, ketoconazole, voriconazole, and fluconazole at minimum inhibitory concentration (MIC) against planktonic form and at 10× MIC and 50× MIC on the biomass and metabolic activity of these biofilms. Biofilm structure was analyzed by optical microscopy using Congo-red staining, confocal and scanning electron microscopy. Strains were classified for biofilm-forming ability, through the analysis of absorbance of crystal violet retained by biomass of mature biofilms. We found that all S. brasiliensis (n = 10), S. schenckii sensu stricto (n = 2), S. globosa (n = 2), and S. mexicana (n = 4) strains were strong biofilm-producers. The analyzed biofilms had dense network of hyphae and conidia immersed in extracellular matrix, with presence of water channels. Antifungal drugs at the three tested concentrations showed different effects on biomass and metabolic activity of biofilms. However, the best inhibitory response was observed with 50× MIC of amphotericin B and caspofungin, which reduced these parameters. Furthermore, high drug concentrations, especially amphotericin B and caspofungin, showed antifungal activity against these biofilms, probably because they damaged the architecture and extracellular matrix, allowing diffusion of the drugs.

Rapid detection of fluconazole resistance in Candida tropicalis by MALDI-TOF MS.

Abstract: With the changing epidemiology and emergence of antifungal resistance among Candida species, rapid antifungal susceptibility testing (AFST) is crucial for optimization of antifungal therapy. This study was conducted to standardize a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI -TOF MS) based AFST method (ms-AFST) for susceptibility of Candida tropicalis isolates. Clinical isolates of C. tropicalis were confirmed for fluconazole resistance by the CLSI (M27-A3) method. The incubation period and drug concentration were optimized to determine the minimal profile change concentration (MPCC) by MALDI-TOF MS. The data were analyzed first by direct visual observation of the spectra followed by composite correlation index (CCI) matrix analysis, virtual gel analysis, and cluster analysis for confirmation. Finally, the correlation between minimum inhibitory concentrations (MICs) and MPCCs was evaluated. A total of 15 fluconazole resistant (MICs ranging from 16 to 128 μg/ml) and 19 fluconazole susceptible C. tropicalis isolates (MIC ≤1 μg/ml) were included in this study. All C. tropicalis isolates had significant spectral changes after 4h incubation with fluconazole. Of 34 isolates, MPCCs and MICs were equivalent for 16 isolates, and the MPCC was one dilution lower than the respective MIC in the remaining 18 isolates. This finding was further supported by visual analysis, CCI matrix analysis, virtual gel and principal component analysis dendrogram analysis. The correlation between MPCC and MIC was significant (P < .05). Therefore, a MALDI-TOF MS based AFST assay may be used as a rapid screening technique for fluconazole resistance in C. tropicalis.

Searching for clues for eighteen years: Deciphering the ecological determinants of Cryptococcus gattii on Vancouver Island, British Columbia.

Abstract: Cryptococcus gattii emerged on Vancouver Island in 1999 for unknown reasons, causing human and animal fatalities and illness. The apparent emergence of this fungus in another temperate area, this time in the Pacific Northwest, suggests the fungus may have expanded its ecological niche. Yet studies that directly examine the potential roles of climatic and land use changes on C. gattii are still lacking. We aim to summarize the existing global literature on the ecology of C. gattii, with particular focus on the gap in knowledge surrounding the potential effects of climatic and land use changes. We systematically reviewed English peer-reviewed literature on the ecological determinants of C. gattii. We included studies published from January 1970 through June 2016 and identified 56 relevant studies for our review. We identified environmental isolations of C. gattii from 18 countries, spanning 72 separate regions across six continents. Fifty-three tree species were associated with C. gattii, spanning 10 climate classifications and 36 terrestrial ecoregions. No studies directly tested the potential effects of climatic changes (including climatic oscillations and global climate change) on C. gattii, while only one study directly assessed those of land use change. To improve model predictions of current and future distributions of C. gattii, more focus is needed on the potential effects of climatic and land use changes to help decrease the public health risk. The apparent emergence of C. gattii in British Columbia is also an opportunity to explore the factors behind emerging infectious diseases in Canada and elsewhere.

Molecular epidemiology of a Malassezia pachydermatis neonatal unit outbreak.

Abstract: The non-lipid-dependent yeast Malassezia pachydermatis is predominantly zoophilic but occasionally colonizes the human skin. This yeast caused an outbreak in a neonatal iIntensive care unit (NICU). This study aimed to describe the molecular epidemiology of this M. pachydermatis outbreak. All the M. pachydermatis isolates collected at a French University Hospital from January 2012 to April 2013 were included in the study. M. pachydermatis isolates, sampled from various biological samples sites in 25 patients, were identified via MALDI-TOF mass spectrometry and typed using intergenic-spacer 1 (IGS1) nucleotide sequence polymorphisms analysis. By analyzing 90 IGS1 sequences (including 43 deposited in GenBank), we found that of the 186 M. pachydermatis isolates, 47 were viable for typing and all of them clustered within type 3; 78.7% clustered within the 3D subtype; the remaining clustered within three newly described subtypes: 3E (4.3%), 3F (8.5%) and 3 G (8.5%). No particular subtype was associated with a collection site or a particular time period. This first molecular investigation of a M. pachydermatis outbreak in neonates showed that multiple genotypes can colonize the same neonate patient by. The source of this polyclonal outbreak could not be identified. It stopped after infection control measures, including the prohibition of a lipid-rich moisturizing hand cream used by the health care staff, had been implemented.

Yeast identification by sequencing, biochemical kits, MALDI-TOF MS and rep-PCR DNA fingerprinting

Abstract: No study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI-TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, using 71 clinically important yeast isolates (excluding Candida albicans) collected from a single centre, we determined the concordance of 28S nrDNA and ITS sequencing and evaluated the performance of two commercial test kits, two MALDI-TOF MS platforms, and rep-PCR DNA fingerprinting. 28S nrDNA and ITS sequencing showed complete agreement on the identities of the 71 isolates. Using sequencing results as the standard, 78.9% and 71.8% isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively; and 90.1% and 80.3% isolates were correctly identified using the Bruker and Vitek MALDI-TOF MS platforms, respectively. Of the 18 strains belonging to the Candida parapsilosis species complex tested by DiversiLab automated rep-PCR DNA fingerprinting, all were identified only as Candida parapsilosis with similarities ≥93.2%, indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, hierarchical cluster analysis of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters, indicating that this technology can potentially differentiate the three species. To achieve higher accuracies of identification, the databases of commercial biochemical test kits, MALDI-TOF MS platforms, and DiversiLab automated rep-PCR DNA fingerprinting needs further enrichment, particularly for uncommonly encountered yeast species.

Black yeasts in the omics era: Achievements and challenges.

Abstract: Black yeasts (BY) comprise a group of polyextremotolerant fungi, mainly belonging to the order Chaetothyriales, which are capable of colonizing a wide range of extreme environments. The tolerance to hostile habitats can be explained by their intrinsic ability to survive under acidic, alkaline, and toxic conditions, high temperature, low nutrient availability, and osmotic and mechanical stress. Occasionally, some species can cause human chromoblastomycosis, a chronic subcutaneous infection, as well as disseminated or cerebral phaeohyphomycosis. Three years after the release of the first black yeast genome, the number of projects for sequencing these organisms has significantly increased. Over 37 genomes of important opportunistic and saprobic black yeasts and relatives are now available in different databases. The whole-genome sequencing, as well as the analysis of differentially expressed mRNAs and the determination of protein expression profiles generated an unprecedented amount of data, requiring the development of a curated repository to provide easy accesses to this information. In the present article, we review various aspects of the impact of genomics, transcriptomics, and proteomics on black yeast studies. We discuss recent key findings achieved by the use of these technologies and further directions for medical mycology in this area. An important vehicle is the Working Groups on Black Yeasts and Chromoblastomycosis, under the umbrella of ISHAM, which unite the clinicians and a highly diverse population of fundamental scientists to exchange data for joint publications.