Medical Mycology

1. Medical Mycology Dr C. O. Morton Prof. T. R. Rogers Dept of Clinical Microbiology

2. Today’s Lecture • Introduction to Fungi • Mycotoxicology • Fungal Cell Wall • Fungal Diseases • Diagnosis • Treatment • Reading List

3. Fungi are Eukaryotic, spore-bearing, heterotrophic organisms that produce extracellular enzymes and absorb their nutrients. Fungi – Kingdom Mycota Divisions - Phyla – Chytridiomycotina, Glomeromycota, Zygomycota, Basidiomycota, Ascomycota Microsporidia, Neocallimastigomycota

4. Fungi are Everywhere In Medicine Food Spoilage Superficial Colonisation

5. Yeast Hyphae Fungi • Of the 50-250,000 fungal species less than 200 cause human disease and only a dozen or so on a regular basis • Yeasts: unicellular fungi reproduce by budding • • Moulds (filamentous): produce hyphae and mycelium • • Dimorphic: grow as moulds (environment) or yeasts (in human host)

6. Basic Structure Woronin body Hyphal Tip

7. Aspergillus (Anamorph) Mitosporic State Neosartorya (Teleomorph) Sexual State

8. The Fungal Cell Wall Rigid structure surrounding the cell

9. Cell wall • The fungal cell wall is essential for growth and viability • Pathogen associated molecular patterns (PAMPS) – the immune systems of most organisms recognise fungal cell wall components such as ß-d-glucan and mannans • Melanin – is an important component of fungal cells walls especially in spores. It protects against UV radiation and Reactive Oxygen Species • Composition can affect action of antimicrobial agents – Candida mutants lacking mannosylphosphate in their cell wall displayed enhanced resistance to cationic antimicrobial peptides via reduced peptide binding (Harris et al. 2009)

10. Immune Avoidance • Cryptococcus neoformans – produces a polysaccharide capsule – possible functions in avoiding phagocytosis, affects antibodies – capsule deficient mutants are less virulent (Perfect, 2005) • Hydrophobin layer of Aspergillus conidia renders them inert to the immune system (Aimanianda et al., 2009)

11. Fungi Need to Eat • Heterotrophic • Secrete extracellular enzymes • Absorptive nutrition (Osmotrophs) • Saprobes: decay dead organic matter • Pathogens: biotroph, necrotroph • Symbionts: parasites - commensals - mutualists

12. Fungi Interact with other organisms • Fungi may gain nutrients through parasitism of other organisms Plant Parasitism “The Frog problem” Chytridiomycosis Affecting 30% of the amphibian species of the world?? Nematode Parasitism

13. Mycotoxicosis

14. Mycotoxins • Mycotoxins are low-molecular-weight secondary metabolites of fungi • Often produced by food spoilage organisms or in basidiocarps (Mushrooms) • mycotoxins are an important chronic dietary risk factor • Aflatoxins - Aspergillus spp.; Citrinin – Penicillium spp.; Ergot Alkaloids – Claviceps spp. – Ergotism; Fuminosins – Fusarium spp.

15. Genus/Species: Amanita muscaria • Slide Reference #: GK 016 • Image Type:Macroscopic Morphology (Basidiocarps) • Disease(s): Mycotoxicosis Amanita muscaria http://www.doctorfungus.org

16. Genus/Species: Amanita phalloides • Slide Reference #: GK 017 • Image Type:Macroscopic Morphology (Basidiocarp) • Disease(s): Mycotoxicosis Amanita phalloides http://www.doctorfungus.org

17. Fungal Spores

18. Humans and Fungi • Humans present a series of diverse microenvironments and barriers to nutrient acquisition, including: • pH – human body has wide pH range – fungi prefer acidic conditions • Temperature – 37 °C is inhospitable to many fungi • Nutritional immunity – sequestration of essential micronutrients such as iron • Physical barriers – skin is composed of polymers that many fungi cannot degrade • Gaseous tension – the ration of O2 to CO2 varies between the surface and within tissue

19. Host immune system • Innate immune system • Pamps, prr • Tlr dectin (segal)

20. Primary Route of Infection (Ebel, F.)

21. Immune System and Fungal Infection Segal (2009 )

22. Fungi That Cause Human Infection • Yeast: Candida albicans, Cryptococcus neoformans • Mould: Aspergillus, Penicillium, Fusarium, Scedosporium • Dimorphic: Histoplasma capsulatum • Associated with Fungi • Sick building syndrome • “Curse of the Pharaohs”

23. Classification of human fungal infections • Superficial: ringworm (dermatophytes), thrush (Candida species), dandruff (Pityrosporum) • Subcutaneous: involve the dermis of the skin, deep tissues or bone. Usually found in tropics/sub-tropics where caught walking barefoot eg, mycetoma • Systemic: due to pathogenic (Histoplasma) or opportunistic (Aspergillus) fungi

25. Dermatophytosis • Tinea – describes an infection caused by a dermatophyte (ringworm fungi) • Specialised pathogenic fungi • Caused by – Trichophyton, Microsporum, Epidermophyton • Worldwide distribution • Key feature is keratin degradation • Don’t tend to grow at 37 °C (Brasch 2008)

26. Dermatophytes Onychomycosis Tinea pedis Tinea corporis

27. Invasive Fungal Disease

28. Invasive Fungal Infection • Invasive fungal infections are major causes for morbidity and mortality in severely ill or immunocompromised patients • Main causative agents are Candida spp, Aspergillus spp. • Emerging infectious fungi – Fusarium spp. Scedosporium spp, Zygomycetes, e.g. Mucor spp • Invasive aspergillosis (IA) occurs in 10% of patients undergoing haematopoietic stem cell transplantation (HSCT) and 25% in autopsy of leukaemic patients • IA has reported mortality rates of 86% and 66% for pulmonary and sinus IA

29. Candida spp • Is usually a harmless commensal organism • It causes both superficial and invasive infections where the host is immunocompromised or epithelial barriers have been damaged • Candida albicans – primary cause of candidiasis • Aspects of virulence • Dimorphism • Phenotypic and mating type switching • Biofilms – higher resistance to antifungals

30. Candida spp can cause disseminated infection

31. Aspergillus fumigatus (Neosartorya fumigata) Ubiquitous soil microbe Decay of organic matter in compost heaps Dispersed by spores, conidia. Opportunistic pathogen of mammals and birds Most important cause of Invasive Fungal Disease in Immunocompromised individuals

32. Aspergillus fumigatus In a tissue sample conidia germination hyphae mycelium

33. Mycetoma • Is most common in Africa and South America • Is a chronic destructive disease affecting skin, underlying tissue and sometimes adjacent bone • Caused by various fungi including Madurella spp., Scedosporium spp., Leptosphaeria spp. • Infection results from traumatic implantation of spores into the skin, e.g. thorns, splinters http://www.doctorfungus.org • Legend:Multiple draining sinuses, swollen tissue, and sclerotia are present. Genus/Species:Madurella mycetomatis Image Type:Clinical Presentation

34. Histoplasmosis • The most common endemic mycosis in North America, also found in Central and South America • A thermally dimorphic fungus, found as a mould in the environment but as budding yeast in tissue • Inhalation of spores is the primary route of infection • Prolonged exposure to aerosolised spores is a major risk factor • Fewer than 5% of individuals exposed to the fungus develop symptomatic disease http://www.doctorfungus.org • Genus/Species: Histoplasma capsulatum var. duboisii • Image Type:Histopathology

35. Blastomycosis • Occurs in North and South America, also Africa • It is a mould in the environment but forms large budding yeast in tissues • Infection through inhalation • Normally in individuals with outdoor occupations • The skin is the most common site of disseminated disease http://www.doctorfungus.org • Genus/Species: Blastomyces dermatitidis • Image Type:Histopathology

36. Diagnosis of Fungal Infection • Microscopy – direct staining of fungi in sections can distinguish between yeasts and molds • Culture – can lead to diagnosis of the exact species. Candida can be grown in blood cultures but Aspergillus cannot • Serology – direct detection of fungal antigens in serum samples. ELISA to detect galactomannan (Platelia – BioRad) or detection of ß-d-glucan, does not detect Cryptococcus spp or zygomycetes • Radiography – direct observation of patients to spot characteristic signs of infection, e.g. halo signs, cavities • PCR – assays target fungal ribosomal operon, nucleic acid extraction from blood or BAL. Potentially very sensitive but still no standardised tests (Hope et al. 2005)

37. Antifungals

38. Antifungal Agents

39. Antifungal Drug Resistance • Efflux pumps in fungi of the ABC and MFS superfamilies and variation in target genes (e.g. cyp51A) are involved in resistance to azoles • Resistance to polyenes is uncommon. Resistant isolates of rare Candida spp show altered levels of membrane sterols • Resistance to FTC is associated with reduce uptake and reduced activity of genes responsible for conversion of FTC to FUMP; FUMP disrupts RNA synthesis • Mutations in the FKS gene (target) can lead to resistance to echinocandins or resistance may be induced by cell wall salvage mechanisms….

40. Reading • General Mycology Books • Introduction to Modern Mycology by J.W. Deacon • The Fungi by M.J. Carlile, S.C. Watkinson and G.W. Gooday • Review Articles • Cooney, N. M. & Klein, B. S. (2008). Fungal adaptation to the mammalian host: it is a new world, after all. Curr Opin Microbiol11, 511-516. • Hope, W. W., Walsh, T. J. & Denning, D. W. (2005). Laboratory diagnosis of invasive aspergillosis. Lancet Infect Dis5, 609-622. • Segal, B. H. (2009). Aspergillosis. N Engl J Med360, 1870-1884. • Websites • http://www.doctorfungus.org/ • http://www.aspergillus.org.uk/