General properties of fungi

For undergraduate medical students

1. General properties of fungi Dr. Pendru Raghunath Reddy Assistant Professor of Microbiology Dr. VRK Women’s Medical College

2. Introduction Mykes (Greek word) : Mushroom Fungi are eukaryotic protista ; differ from bacteria and other prokaryotes. Cell walls containing chitin (rigidity & support), mannan & other polysaccharides Cytoplasmic membrane contains ergosterols Possess true nuclei with nuclear membrane & paired chromosomes Cytoplasmic contents include mitochondria and endoplasmic reticulum Divide asexually, sexually or by both Unicellular or multicellular Most fungi are obligate or facultative aerobes

3. Cell wall consists of chitin not peptidoglycan like bacteria Thus fungi are insensitive to antibiotics as penicillins Chitin is a polysaccharide composed of long chain of n- acetylglucosamine . Also the fungal cell wall contain other polysaccharide, β - glucan , which is the site of action of some antifungal drugs. Cell membrane consist of ergosterol rather than cholesterol like bacterial cell membrane Ergosterol is the site of action of antifungal drugs, amphtericin B & azole group

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5. Diverse group of heterotrophs. Many are ecologically important saprophytes (consume dead and decaying matter) Others are parasites. Most are multicellular, but yeasts are unicellular. Most are aerobes or facultative anaerobes. Cell walls are made up of chitin (polysaccharide). Over 100,000 fungal species identified. Only about 100 are human or animal pathogens. Most human fungal infections are nosocomial and/or occur in immunocompromised individuals (opportunistic infections).

6. In addition to those species which are generally recognized as pathogenic to man it is firmly established that under unusual circumstances of abnormal susceptibility of patient, or the traumatic implantation of the fungus, other fungi are capable of causing lesions. Those are called (Opportunistic fungi)

7. Predisposing factors Use of Antibiotics, Use of steroids, A debilitating condition of the host, as Diabetes. A concurrent disease such as leukaemia Immunosuppressive conditions 8

8. Fungal Morphology Molds Yeasts Many pathogenic fungi are dimorphic , forming hyphae at ambient temperatures but yeasts at body temperature. 7/21/2013 Dr.T.V.Rao MD 9

9. Understanding the Structure of Fungus Simplest fungus :- Unicellular budding yeast Hypha :- Elongation of apical cell produces a tubular, thread like structure called hypha Mycelium :- Tangled mass of hyphae is called mycelium. Fungi producing mycelia are called molds or filamentous fungi. Hyphae may be septate or non- septate ( coenocytic hyphae )

10. Mycelium Mass of branching intertwined hyphae Vegetative Aerial

11. Vegetative types Favic chandeliers Nodular organs Racquet hyphae Spiral hyphae

12. Classification of fungi Morphological classification Systematic classification

13. Yeasts Yeast-like fungi Filamentous fungi (molds) 4. Dimorphic fungi Morphological classification

14. Yeasts These occur in the form of round or oval bodies which reproduce by the formation of buds known as blastospores Yeasts colonies resemble bacterial colonies in appearance and in consistency The only pathogenic yeast in medical mycology is Cryptococcus neoformans

15. Yeast colonies Mucoid colonies Cryptococcus neoformans

16. Cryptococcus neoformans SEM India Ink Capsule Downloded from www.pharmacy123.blogfa.com

17. Yeast-Like 1.These are fungi which occur in the form of budding yeast-like cells and as chains of elongated unbranched filamentous cells which present the appearance of broad septate hyphae. these hyphae intertwine to form a pseudomycelium . 2. The yeast like fungi are grouped together in the genus Candida .

18. Candida Colonies Candida albicans

19. Candida albicans SEM

20. Filamentous Fungi The basic morphological elements of filamentous fungi are long branching filaments or hyphae , which intertwine to produce a mass of filaments or mycelium Colonies are strongly adherent to the medium and unlike most bacterial colonies cannot be emulsified in water The surface of these colonies may be velvety , powdery , or may show a cottony aerial mycelium.

21. mycelium : septate mycelium : non septate

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23. Colony Morphology

24. Dimorphic Fungi These are fungi which exhibit a yeast form in the host tissue and in vitro at 37 C on enriched media and mycelial form in vitro at 25 C Examples: Histoplasma capsulatum Blastomyces dermatitidis Coccidioides immitis Paracoccidoides brasiliesis Penicillium marneffei Sporothrix schenckii

25. Histoplasma capsulatum - Dimorphism Filamentous mold in environment Thin septate hyphae, microconidia, and tuberculate macroconidia (8-14 µm) Budding yeast (2-4 µm) in tissue Dimorphic transition is thermally dependent and reversible (25°C  37°C). Hyphae , micro- and macroconidia Yeast within histiocyte

26. Systematic classification Based on sexual spores formation: 4 classes Zygomycetes Ascomycetes reproduce sexually Basidiomycetes Deuteromycetes (fungi imperfectii )

27. Zygomycetes Lower fungi Broad, nonseptate hyphae Asexual spores - Sporangiospores : present within a swollen sac- like structure called Sporangium Examples: Rhizopus , Absidia , Mucor  

28. Ascomycetes Sexual spores called ascospores are present within a sac like structure called Ascus . Each ascus has 4 to 8 ascospores Includes both yeasts and filamentous fungi

29. Ascomycetes Narrow, septate hyphae Asexual spores are called conidia borne on conidiophore Examples: Penicillium , Aspergillus

30. Basidiomycetes Sexual fusion results in the formation of a club shaped organ called base or basidium which bearspores called basidiospores Examples: Cryptococcus neoformans , mushrooms

31. Deuteromycetes or Fungi imperfectii Group of fungi whose sexual phases are not identified Grow as molds as well as yeasts Most fungi of medical importance belong to this class Examples: Coccidioides immitis , Paracoccidioides brasiliensis , Candida albicans

32. Reproduction and sporulation Types of fungal spores Sexual spores Asexual spores

33. Sexual spores Sexual spore is formed by fusion of cells and meiosis as in all forms of higher life Ascospores Ascus Ascocarp Basidiospores Zygospores

34. Asexual spores These spores are produced by mitosis Vegetative spores Aerial spores

35. Vegetative spores Blastospores : These are formed by budding from parent cell, as in yeasts Arthrospores – formed by segmentation & condensation of hyphae Chlamydospores – thick walled resting spores e.g. C.albicans

36. Aerial spores 1. Conidiospores Spores borne externally on sides or tips of hyphae are called conidiospores or simply conidia 2. Microconidia 3. Macroconidia 4. Sporangiospores

37. Microconidia - Small, single celled Macroconidia – Large and septate and are often multicellular

38. Immunity to fungal diseases The defense to fungal infection involves both innate and aquired immunity The passive protection is provided by intact skin and mucosal surfaces The fatty acids like sebum also provide protection because of their antifungal activity The alveolar macrophages are important in engulfing cells in lungs which are removed by ciliary action and coughing A variety of innate defense factors in saliva such as lysozymes and lactoferrin contribute to mucosal protection

39. Cellular immunity For the most of fungi, cellular immunity is mainstay of host defenses Monocyte -macrophage activity, especially in respiratory tract, appears to be major defence mechanism against most fungi Renal transplant recipients have peculiar predilection for cryptococcal infection Humoral immunity The precise role of humoral immunity in host defense against fungal infections is difficult to determine

40. Diagnosis of fungal diseases Clinical diagnosis Laboratory diagnosis

41. Clinical diagnosis The clinical criteria may give presumptive diagnosis of fungal infections The superficial and subcutaneous mycoses often produce characteristic lesions that strongly suggest their fungal etiology In case of systemic mycoses, there is no sign or symptom that specifically suggests a fungal disease

42. Laboratory diagnosis Specimens collection Respiratory specimens CSF Blood Tissue, Bone marrow and body fluids Skin, hair and nail Urine, stool Vaginal secretions Corneal scrapings

43. Direct examination The direct demonstration of fungal elements is essential in establishing diagnosis by detecting presence of causative agent in the clinical material The following procedures are adopted to directly observe fungi in clinical specimens Wet mounts Histopathology Fluorescent-antibody technique Gram staining

44. Wet mounts KOH wet mount Slide KOH Most of the specimens can be examined in wet mounts after partial digestion with 10-20% KOH The clinical specimens like skin, hair and nails should be mounted under cover slip in KOH on slide This clears material within 5 – 20 minutes, depending on its thickness A slight warming over a low flame hastens digestion of keratin

45. KOH can also be supplemented with DMSO to increase clearing of fungi especially in skin scrapings and nail clippings The KOH can be supplemented with a fluorescent dye, calcofluor white (CFW) The CFW supplemented KOH especially in corneal scrapings can detect even scanty amount of fungal elements Tube KOH The tube KOH is prepared mainly for biopsy specimens, which take longer time for dissolution The homogenized biopsy tissue is dissolved in 10% KOH and examined after keeping for an overnight in an incubator at 37 C

46. KOH mount Blastomyces dermatitidis Mold (note: septate hyphae )

47. KOH mount Ectothrix Endothrix

48. Other methods The wet india ink and nigrosin preparations are also very useful in making diagnosis of certain capsulated fungi Histopathology The histopathological examination of biopsy specimens is an excellent way to diagnose mycotic infections The H&E stain is routine procedure in histopathology laboratory and stains most of fungi

49. H&E stain Endospores and sporangia of Rhinosporidium seeberi Cryptococcus neoformans

50. Blastomyces dermatitidis Rhizopus species H&E stain

51. The specific fungal stains, such as periodic acid-Schiff (PAS), Grocott-Gomori’s Methenamine silver (GMS) and Gridely stains are widely used for demonstrating fungi in tissues Mayer’s mucicarmine stain can also be used specifically to show capsular material of Cryptococcus , endospores and sporangia of Rhinosporidium seeberi

52. PAS stain GMS stain

53. Fluorescent-antibody staining May used to detect fungal antigen in clinical material such as pus, blood,CSF , tissue impression smears and in paraffin sections of formalin fixed tissue It is less satisfacory for sputum specimens Detects fungus when there are only few organisms present, as seen in pus from sporotrichosis Its use is limited by restricted availability of specific antisera

54. Gram staining All fungi are gram positive Gram stained smear of Candida species

55. Fungal culture The solid media are employed for fungal culture The medium commonly employed is emmon’s modification of Sabouraud Dextrose Agar (SDA) [pH – 5.4] The media may supplemented with antibiotics, such as gentamicin and chloramphenicol to minimize bacterial contamination and cycloheximide to inhibit saprophytic fungi The special media may be used for isolation and to help rapid identification, when identity of a particular pathogen is strongly suspected Eg : Bird seed agar for Cryptococcus neoformans Potato dextrose agar, Brain heart infusion (BHI) agar and Czapek Dox agar may also be used for isolation of fungi

56. Incubation Cultures are routinely incubated in parallel at room temperature (for weeks) and at 37 C for days Many fungi develop relatively slowly and cultures should be retained for atleast 2-3 weeks (in some cases upto 6 weeks) before being discarded Yeasts usually grow within 1-5 days

57. Interpretation of fungal cultures The significance of fungal isolate depends on its source and identity The isolation of an established pathogen such as H. capsulatum or Coccidioides species from any specimen is generally regarded as an evidence of infection In case of commensal or opportunistic fungi following points should be considered Isolation of same strain in all culture tubes Repeated isolation of same strain in multiple specimens Immune status of patients and Serological evidence to confirm significance of isolate

58. Identification of fungi Gross or macroscopic examination of cultures Microscopic examination Tease mount Slide culture

59. Tease mount For microscopic examination, fragment of fungal culture is teased out using two teasing needles and placed on a glass slide in drop of LCB stain To study the morphology of hyphae , spores and other structures, tease mounts are prepared in lactophenol cotton blue (LCB) which contains lactic acid, phenol, glycerol and cotton blue Microscopic characteristics that should be observed are the following Sepatate versus sparsely septate hyphae Spores or conidia

60. Slide culture Is used to study undisturbed morphological details of fungi, particularly relationship between reproductive structures like conidia, conidiophore and hyphae It is indicated when teased mount of LCB is inconclusive in particular fungal isolate

61. Procedure Place sterile microscopic slide on bent glass rod at the bottom of petri dish A piece of one square centimeter block of cornmeal agar or potato dextrose agar is put up on the slide Inoculate the fungal strain under identification at four sides of agar block Cover the inoculated block with sterile coverslip and incubate at 25 C in BOD incubator

62. Slide culture

63. Add a little sterile distilled water on filter paper to avoid drying of agar When growth appears, place drop of LCB on slide and coverslip from block Cellophane tape preparation A piece of tape is gently laid over portion of fungal colony and slowly lifted and place over a glass slide containing LCB solution This preparation has come into greater use to overcome obstacles of time consumption and requirement of extra equipment to prepare slide culture

64. Aspergillus Penicillium

65. Miscellaneous tests for the identification of molds Hair perforation test Urease test Thiamine requirement Miscellaneous tests for identification of yeasts Germ tube production, carbohydrate assimilation, chromogenic substrates, potassium nitrate assimilation, temperature studies and urease

66. Serologic tests Detection of antibodies Immunodiffusion Countercurrent immunoelectrophoresis (CIE) Whole cell agglutination Complement fixation ELISA Detection of antigens Latex particle agglutination ELISA

67. Molecular techniques PCR DNA probes

68. Mycoses Infection caused by fungus is known as mycosis (Plural mycoses) Classification of mycoses Classification of fungal disease according to primary sites of infections Superficial mycoses Cutaneous mycoses Subcutaneous mycoses Systemic mycoses Opportunistic mycoses