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Next-Generation Sequencing for Hypothesis-Free Genomic Detection
Frickmann et al. BMC Microbiology (2019) 19:75 https://doi.org/10.1186/s12866-019-1448-0 RESEARCH ARTICLE Open Access Next-generation sequencing for hypothesis- free genomic detection of invasive tropical infections in poly-microbially contaminated, formalin-fixed, paraffin-embedded tissue samples – a proof-of-principle assessment Hagen Frickmann1,2* , Carsten Künne3, Ralf Matthias Hagen4, Andreas Podbielski2, Jana Normann2, Sven Poppert5,6, Mario Looso3 and Bernd Kreikemeyer2 Abstract Background: The potential of next-generation sequencing (NGS) for hypothesis-free pathogen diagnosis from (poly-)microbially contaminated, formalin-fixed, paraffin embedded tissue samples from patients with invasive fungal infections and amebiasis was investigated. Samples from patients with chromoblastomycosis (n = 3), coccidioidomycosis (n = 2), histoplasmosis (n = 4), histoplasmosis or cryptococcosis with poor histological discriminability (n = 1), mucormycosis (n = 2), mycetoma (n = 3), rhinosporidiosis (n = 2), and invasive Entamoeba histolytica infections (n = 6) were analyzed by NGS (each one Illumina v3 run per sample). To discriminate contamination from putative infections in NGS analysis, mean and standard deviation of the number of specific sequence fragments (paired reads) were determined and compared in all samples examined for the pathogens in question. Results: For matches between NGS results and histological diagnoses, a percentage of species-specific reads greater than the 4th standard deviation above the mean value of all 23 assessed sample materials was required. Potentially etiologically relevant pathogens could be identified by NGS in 5 out of 17 samples of patients with invasive mycoses and in 1 out of 6 samples of patients with amebiasis. Conclusions: The use of NGS for hypothesis-free pathogen diagnosis from contamination-prone formalin- fixed, paraffin-embedded tissue requires further standardization. -
Uncommon Fungi and Related Species
Uncommon Fungi and 268 Related Species Duane R. Hospenthal SHORT VIEW SUMMARY SCEDOSPORIUM APIOSPERMUM Diagnosis FUSARIUMM SPP. (PSEUDALLESCHERIA BOYDIII) SPECIES Diagnosis is made by culture recovery from the } Definition COMPLEX infected site. } Can cause disseminated infection in } Because L. prolificanss may colonize airways, Definition immunocompromised patients. sputum cultures may not reflect infection. } Infection of the lungs, bones and joints, or } Common cause of keratitis and other eye central nervous system (CNS); may be Therapy infections in contact lens wearers and disseminated. } No effective therapy. Consider voriconazole following trauma. } Also causes mycetoma (see Chapter 261). with amphotericin B. } Skin and soft tissue infection after trauma, onychomycosis; can cause mycetoma. Epidemiology DARK-WALLED FUNGI (BIPOLARIS, } Typically occurs in the immunocompromised or EXOPHIALA, EXSEROHILUM, Epidemiology following trauma. PHIALOPHORA, OCHROCONIS, } Common plant pathogens; found in soil and } CNS infection in immunocompetent persons CURVULARIA, OTHERS) organic debris. after near drowning. Have been recovered in hospital water supplies. Definition } } Organism can be found in soil and fresh water, } This infection involves fungi that have melanin Microbiology especially stagnant or polluted. in their cell walls and may appear dark walled } The most common species infecting humans Microbiology in tissue. belong to one of three species complexes: } Scedosporium apiospermum, Scedosporium } Infection is often termed Fusarium solani, Fusarium oxysporum, or boydiii (formerly Pseudallescheria boydiii), and “phaeohyphomycosis” and typically presents Fusarium fujikuroi, although the number of Scedosporium aurantiacumm are the most as localized skin and soft tissue infections, species identified as causing infection is common species infecting humans. CNS infections, or allergic sinusitis. increasing as molecular methods of } Identification is typically made by DNA } Dark-walled fungi that cause identification have supplanted morphology. -
Introduction to Mycology
INTRODUCTION TO MYCOLOGY The term "mycology" is derived from Greek word "mykes" meaning mushroom. Therefore mycology is the study of fungi. The ability of fungi to invade plant and animal tissue was observed in early 19th century but the first documented animal infection by any fungus was made by Bassi, who in 1835 studied the muscardine disease of silkworm and proved the that the infection was caused by a fungus Beauveria bassiana. In 1910 Raymond Sabouraud published his book Les Teignes, which was a comprehensive study of dermatophytic fungi. He is also regarded as father of medical mycology. Importance of fungi: Fungi inhabit almost every niche in the environment and humans are exposed to these organisms in various fields of life. Beneficial Effects of Fungi: 1. Decomposition - nutrient and carbon recycling. 2. Biosynthetic factories. The fermentation property is used for the industrial production of alcohols, fats, citric, oxalic and gluconic acids. 3. Important sources of antibiotics, such as Penicillin. 4. Model organisms for biochemical and genetic studies. Eg: Neurospora crassa 5. Saccharomyces cerviciae is extensively used in recombinant DNA technology, which includes the Hepatitis B Vaccine. 6. Some fungi are edible (mushrooms). 7. Yeasts provide nutritional supplements such as vitamins and cofactors. 8. Penicillium is used to flavour Roquefort and Camembert cheeses. 9. Ergot produced by Claviceps purpurea contains medically important alkaloids that help in inducing uterine contractions, controlling bleeding and treating migraine. 10. Fungi (Leptolegnia caudate and Aphanomyces laevis) are used to trap mosquito larvae in paddy fields and thus help in malaria control. Harmful Effects of Fungi: 1. -
Fungi Infections
FUNGAL INFECTIONS mycology mycoses fungemia exo-antigen fungal antigenemia biomarker pre-emptive therapy Fungi FUNGI BACTERIA nucleus eukaryotes prokaryotes cell membrane sterols (ergosterol)* - cell wall chitin, mannan, glucan, murein, teichoic acid, chitosan proteins oxygen almost all strict aerobes facultative and obligate aerobes and anaerobes, - Heterotrophs requiring organic carbon source for growth ( biotrophic, saprophyte) - Extracellular enzymes - host defense: cell-mediated immunity (role of antibodies is minor) -> neutrophil phagocytosis and killing Antifungal agents- mode of action - Polyenes (amphotericinB, nystatines, pimarcin) - Azoles (ketokonazole, itraconazole, fluconazole, vericonazole, posaconazole) - Echinocandins (caspofungin, mikafungin, anidulafungin ) - Nucleoside analogs(antimetabolites): (5 fluorocytosine) - Allylamines: (tebinafine) Fungal morphotypes Unicellular form (Yeast) Yeasts spherical or ellipsoid fungal cells reproduce by budding Mycelial form : moulds, dermathophytes Molds hyphal or mycelial form of growth branching filaments (filamentous) . Fungal morphotypes Unicellular form (Yeast) FUNGUS FAMILY YEAST MOLDs & dermatophytes Candida, Cryptococcus, Dymorphic fungi Malessezia, Geotrichum, Aspergillus, Penicillium, Blastomyces, Coccidioides, Trichosporon, Rodotorula Mucor, Rhizopus, Fusarium, Histoplasma, Paracoccidioides etc. Cladosporium, or Scopulariopsis Dimorphic fungi – have two growth forms: molds & yeast, which develope under different growth conitions phaeohyphomycetes Most authorities use the -
10-ELS-OXF Kurtzman1610423 CH002 7..20
Part II Importance of Yeasts Kurtzman 978-0-444-52149-1 00002 Kurtzman 978-0-444-52149-1 00002 Chapter 2 c0002 Yeasts Pathogenic to Humans Chester R. Cooper, Jr. regularly encounter the organisms described below. In fact, many s0010 1. INTRODUCTION TO THE MEDICALLY medical mycologists spend entire careers without direct clinical expo- IMPORTANT YEASTS sure to many of these fungi. Rather, the purpose of this review is to enlighten the non-medical mycologist as to the diversity of yeast and p0010 Prior to global emergence of the human immunodeficiency virus mold species regularly associated with human and animal disease (HIV), which is the causative agent of acquired immunodeficiency that also, at least in part, present a unicellular mode of growth in vivo. syndrome (AIDS), approximately 200 fungal pathogens were recog- The following descriptions present a concise overview of the key p0025 nized from among the more than 100,000 then-known fungal spe- biological and clinical features of these fungi. Where appropriate, refer- cies (Kwon-Chung and Bennett 1992, Rippon 1988). About 50 of ences to recent reviews of particular disease agents and their patholo- these species were regularly associated with fungal disease (myco- gies are provided. For a global perspective of fungal diseases, including sis). Since then, there has been a concurrent dramatic increase in in-depth clinical discussions of specific pathologies, diagnoses, and both the number of known fungal species and the incidence of treatments, the reader is referred to several outstanding and recently mycoses that they cause. Moreover, the spectrum of pathogenic fungi published texts (Anaissie et al. -
FUNGI Why Care?
FUNGI Fungal Classification, Structure, and Replication -Commonly present in nature as saprophytes, -transiently colonising or etiological agenses. -Frequently present in biological samples. -They role in pathogenesis can be difficult to determine. Why Care? • Fungi are a cause of nosocomial infections. • Fungal infections are a major problem in immune suppressed people. • Fungal infections are often mistaken for bacterial infections, with fatal consequences. Most fungi live harmlessly in the environment, but some species can cause disease in the human host. Patients with weakened immune function admitted to hospital are at high risk of developing serious, invasive fungal infections. Systemic fungal infections are a major problem among critically ill patients in acute care settings and are responsible for an increasing proportion of healthcare- associated infections THE IMPORTANCE OF FUNGI • saprobes • symbionts • commensals • parasites The fungi represent a ubiquitous and diverse group of organisms, the main purpose of which is to degrade organic matter. All fungi lead a heterotrophic existence as saprobes (organisms that live on dead or decaying matter), symbionts (organisms that live together and in which the association is of mutual advantage), commensals (organisms living in a close relationship in which one benefits from the relationship and the other neither benefits nor is harmed), or as parasites (organisms that live on or within a host from which they derive benefits without making any useful contribution in return; in the case of pathogens, the relationship is harmful to the host). Fungi have emerged in the past two decades as major causes of human disease, especially among those individuals who are immunocompromised or hospitalized with serious underlying diseases. -
Introduction to Bacteriology and Bacterial Structure/Function
INTRODUCTION TO BACTERIOLOGY AND BACTERIAL STRUCTURE/FUNCTION LEARNING OBJECTIVES To describe historical landmarks of medical microbiology To describe Koch’s Postulates To describe the characteristic structures and chemical nature of cellular constituents that distinguish eukaryotic and prokaryotic cells To describe chemical, structural, and functional components of the bacterial cytoplasmic and outer membranes, cell wall and surface appendages To name the general structures, and polymers that make up bacterial cell walls To explain the differences between gram negative and gram positive cells To describe the chemical composition, function and serological classification as H antigen of bacterial flagella and how they differ from flagella of eucaryotic cells To describe the chemical composition and function of pili To explain the unique chemical composition of bacterial spores To list medically relevant bacteria that form spores To explain the function of spores in terms of chemical and heat resistance To describe characteristics of different types of membrane transport To describe the exact cellular location and serological classification as O antigen of Lipopolysaccharide (LPS) To explain how the structure of LPS confers antigenic specificity and toxicity To describe the exact cellular location of Lipid A To explain the term endotoxin in terms of its chemical composition and location in bacterial cells INTRODUCTION TO BACTERIOLOGY 1. Two main threads in the history of bacteriology: 1) the natural history of bacteria and 2) the contagious nature of infectious diseases, were united in the latter half of the 19th century. During that period many of the bacteria that cause human disease were identified and characterized. 2. Individual bacteria were first observed microscopically by Antony van Leeuwenhoek at the end of the 17th century. -
Sporothrix Schenckii: ➢ Thermal Dimorphic
Subcutaneous mycoses ➢1-Mycetoma ➢2-Sporotrichosis ➢3-Chromoblastomycosis ➢4-Rhinosporidiosis ➢5- Lobomycosis ➢6- Entomophthoramycosis Sporotrichosis Rose gardener’s disease Chronic desease Agent Sporothrix schenckii: ➢ Thermal dimorphic ➢ In soil ➢ On decaying vegetation,plants,plant products (hay, straw, sphagnum moss), and a variety of animals (cats) ➢ less than 37° C (hyphal) ➢ 37° C (yeast) ➢ Sporothrix brasiliensis ➢ Sporothrix globosa Epidemiology ➢Worldwide ➢Tropical regions ➢Mexico ➢Brazile ➢France ➢USA Occupational disease: ➢Farmers ➢ ➢Workers ➢Gardeners ➢Florists Predisposing factors: ➢Trauma ➢Inhalation (very rarely) ➢HIV Clinical Syndromes 1-lymphocutaneous 2-Fixed cutaneous 3- Osteoarticular involvement 4-Pulmonary 5-Systemic Primary infection 1-Lymphocutaneous sporotrichosis 2-Fixed cutaneous sporotrichosis: Fixed cutaneous sporotrichosis verrucous-type sporotrichosis localized cutaneous type Paronychia sporotrichosis Osteoarticular involvement Pulmonary sporotrichosis: ➢Alcoholic ➢Pulmonary tuberculosis, diabetes mellitus and steroid ➢A productive cough ➢Low-grade fever ➢Weight loss Systemic sporotrichosis Transmission: ➢Dog bite ➢parrot bite ➢Insects bite ➢Cases of animal-to-human transmission Laboratory Diagnosis: 1-Collection of samples: ➢Drainage from skin lesions ➢Exudates ➢Pus ➢Blood ➢Pulmonary secretions ➢Tissue biopsy specimens 2-Direct examination ➢Gram ➢PAS ➢GMS ➢H & E ❖Yeast Cells ❖Asteroid body: Elongated Buds (“Cigar Body”) Wet Mount BHI Blood 37˚C Yeast with Elongated Daughter Cell Biopsy of subcutaneous tissue -
Black Fungal Extremes
Studies in Mycology 61 (2008) Black fungal extremes Edited by G.S. de Hoog and M. Grube CBS Fungal Biodiversity Centre, Utrecht, The Netherlands An institute of the Royal Netherlands Academy of Arts and Sciences Black fungal extremes STUDIE S IN MYCOLOGY 61, 2008 Studies in Mycology The Studies in Mycology is an international journal which publishes systematic monographs of filamentous fungi and yeasts, and in rare occasions the proceedings of special meetings related to all fields of mycology, biotechnology, ecology, molecular biology, pathology and systematics. For instructions for authors see www.cbs.knaw.nl. EXECUTIVE EDITOR Prof. dr Robert A. Samson, CBS Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] LAYOUT EDITOR S Manon van den Hoeven-Verweij, CBS Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Kasper Luijsterburg, CBS Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] SCIENTIFIC EDITOR S Prof. dr Uwe Braun, Martin-Luther-Universität, Institut für Geobotanik und Botanischer Garten, Herbarium, Neuwerk 21, D-06099 Halle, Germany. E-mail: [email protected] Prof. dr Pedro W. Crous, CBS Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Prof. dr David M. Geiser, Department of Plant Pathology, 121 Buckhout Laboratory, Pennsylvania State University, University Park, PA, U.S.A. 16802. E-mail: [email protected] Dr Lorelei L. Norvell, Pacific Northwest Mycology Service, 6720 NW Skyline Blvd, Portland, OR, U.S.A. -
Indoor Wet Cells As a Habitat for Melanized Fungi, Opportunistic
www.nature.com/scientificreports OPEN Indoor wet cells as a habitat for melanized fungi, opportunistic pathogens on humans and other Received: 23 June 2017 Accepted: 30 April 2018 vertebrates Published: xx xx xxxx Xiaofang Wang1,2, Wenying Cai1, A. H. G. Gerrits van den Ende3, Junmin Zhang1, Ting Xie4, Liyan Xi1,5, Xiqing Li1, Jiufeng Sun6 & Sybren de Hoog3,7,8,9 Indoor wet cells serve as an environmental reservoir for a wide diversity of melanized fungi. A total of 313 melanized fungi were isolated at fve locations in Guangzhou, China. Internal transcribed spacer (rDNA ITS) sequencing showed a preponderance of 27 species belonging to 10 genera; 64.22% (n = 201) were known as human opportunists in the orders Chaetothyriales and Venturiales, potentially causing cutaneous and sometimes deep infections. Knufa epidermidis was the most frequently encountered species in bathrooms (n = 26), while in kitchens Ochroconis musae (n = 14), Phialophora oxyspora (n = 12) and P. europaea (n = 10) were prevalent. Since the majority of species isolated are common agents of cutaneous infections and are rarely encountered in the natural environment, it is hypothesized that indoor facilities explain the previously enigmatic sources of infection by these organisms. Black yeast-like and other melanized fungi are frequently isolated from clinical specimens and are known as etiologic agents of a gamut of opportunistic infections, but for many species their natural habitat is unknown and hence the source and route of transmission remain enigmatic. Te majority of clinically relevant black yeast-like fungi belong to the order Chaetothyriales, while some belong to the Venturiales. Propagules are mostly hydro- philic1 and reluctantly dispersed by air, infections mostly being of traumatic origin. -
Rhinosporidium Seeberi: a Human Pathogen from a Novel Group of Aquatic Protistan Parasites
Research Rhinosporidium seeberi: A Human Pathogen from a Novel Group of Aquatic Protistan Parasites David N. Fredricks,*† Jennifer A. Jolley,* Paul W. Lepp,* Jon C. Kosek,† and David A. Relman*† *Stanford University, Stanford, California, USA; and †Veterans Affairs, Palo Alto Health Care System, Palo Alto, California, USA Rhinosporidium seeberi, a microorganism that can infect the mucosal surfaces of humans and animals, has been classified as a fungus on the basis of morphologic and histochemical characteristics. Using consensus polymerase chain reaction (PCR), we amplified a portion of the R. seeberi 18S rRNA gene directly from infected tissue. Analysis of the aligned sequence and inference of phylogenetic relationships showed that R. seeberi is a protist from a novel clade of parasites that infect fish and amphibians. Fluorescence in situ hybridization and R. seeberi-specific PCR showed that this unique 18S rRNA sequence is also present in other tissues infected with R. seeberi. Our data support the R. seeberi phylogeny recently suggested by another group. R. seeberi is not a classic fungus, but rather the first known human pathogen from the DRIPs clade, a novel clade of aquatic protistan parasites (Ichthyosporea). Rhinosporidiosis manifests as slow-growing, that has been difficult to classify. Recently, tumorlike masses, usually of the nasal mucosa or R. seeberi has been considered a fungus, but it was ocular conjunctivae of humans and animals. originally thought to be a protozoan parasite (2). Patients with nasal involvement often have Its morphologic characteristics resemble those of unilateral nasal obstruction or bleeding due to Coccidioides immitis: both organisms have polyp formation. The diagnosis is established by mature stages that consist of large, thick-walled, observing the characteristic appearance of the organism in tissue biopsies (Figure 1). -
Therapies for Common Cutaneous Fungal Infections
MedicineToday 2014; 15(6): 35-47 PEER REVIEWED FEATURE 2 CPD POINTS Therapies for common cutaneous fungal infections KENG-EE THAI MB BS(Hons), BMedSci(Hons), FACD Key points A practical approach to the diagnosis and treatment of common fungal • Fungal infection should infections of the skin and hair is provided. Topical antifungal therapies always be in the differential are effective and usually used as first-line therapy, with oral antifungals diagnosis of any scaly rash. being saved for recalcitrant infections. Treatment should be for several • Topical antifungal agents are typically adequate treatment weeks at least. for simple tinea. • Oral antifungal therapy may inea and yeast infections are among the dermatophytoses (tinea) and yeast infections be required for extensive most common diagnoses found in general and their differential diagnoses and treatments disease, fungal folliculitis and practice and dermatology. Although are then discussed (Table). tinea involving the face, hair- antifungal therapies are effective in these bearing areas, palms and T infections, an accurate diagnosis is required to ANTIFUNGAL THERAPIES soles. avoid misuse of these or other topical agents. Topical antifungal preparations are the most • Tinea should be suspected if Furthermore, subsequent active prevention is commonly prescribed agents for dermatomy- there is unilateral hand just as important as the initial treatment of the coses, with systemic agents being used for dermatitis and rash on both fungal infection. complex, widespread tinea or when topical agents feet – ‘one hand and two feet’ This article provides a practical approach fail for tinea or yeast infections. The pharmacol- involvement. to antifungal therapy for common fungal infec- ogy of the systemic agents is discussed first here.