1. MEDICALLY IMPORTANT FUNGI and ANTIFUNGAL THERAPY
DR. BREIDA BOYLE

2. INTRODUCTION
Fungi are a diverse group of sacrophytic and parasitic eukaryotic organisms
Kingdom: Mycota
Of 100,000 fungal species only 100 have pathogenic potential for humans, only a few account for clinically important infections
Mycoses : Human Fungal Diseases
Fungal spores may be important as human allergenic agents

6. INTRODUCTION MYCOSES MUCOSAL: limited to mucosae
CUTANEOUS: limited to the dermis
SUBCUTANEOUS : when infection penetrates significantly beneath the skin
SYSTEMIC : when the infection is deep within the body or disseminated to internal organs

7. PATHOGENIC FUNGI
TRUE
PATHOGENS
OPPORTUNISTIC
PATHOGENS

8. TRUE PATHOGENS Cutaneous infective agents
Subcutaneous infective agents
Actinomadura madurae
Cladosporium
Madurella grisea
Phialophora
Sporothrix schenckii
Epidermophyton species
Microsporum species
Trichophyton species
Systemic infective agents
Blastomyces dermatitidis
Coccidioides immitis
Histoplasma capsulatum
Paracoccidioides brasiliensis

9. OPPORTUNISTIC PATHOGENS
Absidia corymbifera
Aspergillus fumigatus
Candida albicans
Crytococcus neoformans
Pneumocystis carinii
Rhizomucor pusillus
Rhizopus oryzae (R.arrhizus)

10. PATHOGENIC FUNGI
TRUE
PATHOGENS
OPPORTUNISTIC
PATHOGENS

11. CLASSIFICATION OF FUNGI
Depends on :
Characteristic Structures
Habitats
Modes of Growth
Modes of Reproduction
Clinical Setting
DNA Homology

12. Cell Wall and Membrane
Composed mainly of chitin rather than peptidoglycan (bacteria)-so unaffected by antibiotics
Cell Wall also has glucans and Mannans
Chitin: consists of a polymer of N-acetylglucosamine
Fungal Membrane contains ergosterol rather than cholesterol found in mammalian cells, use in antifungal agents such as amphotericin which binds to ergosterolpores that disrupts membrane function cell death

13. Cell Membrane
The imidazole antifungal drugs ( clotrimazole, ketoconazole, miconazole) and the triazole antifungal agents (fluconazole , itraconazole, voriconazole) interact with the C-14 α-demethylase to block demethylation of lansterol to ergosterol, vital component of cell membrane and disruption of it`s synthesis results in death

14. HABITAT
All fungi are heterotrophs ( their require some form of organic carbon for growth)
They depend on transport of soluble nutrients across their cell membrane
To do this they secrete degradative enzymes ( proteases etc) into their immediate environment, therefore they live on dead organic material
So Natural Habitat : is soil or water containing decaying organic matter

15. MODES OF FUNGAL GROWTH UNICELLULAR FILAMENTOUS YEASTS MOLDS
However there are some dimorphic fungi ( they switch between these
Two forms depending on their environment)

16. Filamentous (mold-like) Fungi
Thallus (vegetitive body) –mass of threads with many branches resembling cotton ball
Mass: mycelium
Threads: hyphae, tubular cells that in some fungi are divided into segments –septate whereas in other fungi the hyphae are uninterrupted by crosswalls-nonseptate
Grow by branching and tip elongation

17. YEAST like FUNGI
These fungi exist as populations of single , unconnected , spheroid cells, not unlike many bacteria, although they are sometimes 10 times larger than a typical bacterial cell
Yeasts reproduce by budding
Some fungal species particularly those that cause systemic infection exist as dimorphic fungi

18. REPRODUCTION

19. SPORULATION
The principle way in which fungi reproduce and spread within the environment
Fungal spores are metabolically dormant, protected cells, released by the mycelium in enormous numbers
Borne by the air or water to new sites , where they germinate and establish new colonies
Spores can be generate sexually or asexually

20. ASEXUAL SPORULATION (MITOSIS)
Colour of a particular fungus seen on bread, culture plate is due to the
Conidia, easly airborne and disseminated

21. SEXUAL SPORULATION meiosis
Relatively rare compared to asexual sporulation, and spore shape often
Used as a method of identification

22. CUTANEOUS MYCOSES -DERMATOPHYTOSES
EPIDEMIOLOGY
Three genera-Trichophyton, Epidermophyton, Microsporum
Anthropophilic-reside on the human skin
Zoophilic-reside on the skin of domestic and farm animals
Geophilic-reside in the soil
Transmission from humans or animals is by infected skin scales

23. PATHOLOGY Dermatophytes use keratin as a source of nutrition
Therefore they infect skin, hair, nails
All 3 organisms infect /attack skin, Microsporum does not infect nails and Epidermophyton does not infect hair, they do not invade underlying non-keratinized tissues

24. CLINICAL SIGNIFICANCE
DERMATOPHYTOSES
Characterized by itching,scaling skin patches that can become inflamed and weeping
Infection in different sites may be due to different organisms but is given one name

25. Tinea pedis(Athlete`s foot)
Common organisms are Trichophyton rubrum , Trichophyton mentagrophytes and Epidermophyton floccosum.
Initially between the toes spreads to nails, yellow and brittle
Secondary bacterial infection
Id Reaction

26. Tinea corporis( Ringworm)
Epidermophyton floccosum, Trichophyton, Microsporum
Advancing annular rings with scaly center
Periphery of ring area of active fungal growth, usually inflammed and vesiculated
Non-Hairy areas of trunks mostly

27. Tinea capitis( scalp ringworm)
Trichophyton and Microsporum species
Depends on area
Small scaling patches to involvement of entire hair with hairloss
Microsporum infects hair shafts , Wood`s lamp
More common in children due to medium chain fatty acids(C8-120 in sebum

28. TINEA CRURIS/UNGUIUM
Epidermophyton , Trichophyton rubrum, simliar to ringworm but thighs and genitalia
Trichophyton rubrum, nails thickened discoloured and brittle , Onchomycosis
Treatment for months until all of the infected nail grows out and is trimmed off

29. Tinea vesicolor Pityrasis vesicolor
Due to Malassezia furfur or Pityosporium orbiculare
Treatment , ketoconazole, fluconazole , itraconazole

30. Diagnosis of Dermatophyte Infection
Nail clippings, skin scrapings, Hair /follicile
No role for swabs
Placed in sterile container preferably or between 2 slides
KOH will be added in the lab to dissolve tissue material
Lactophenol blue stain to see if fungal hyphae seen
For full identification culture on selective media required e.g addition of cycloheximide or chloramphenicol, low ph 5.0
May Require days for growth
Macroscopic and microscopic identification of colonies

31. Fungal elements/hyphae

32. T.mentagrophytes

33. T.mentagrophytes

34. Treatment Samples to be sent for fungal staining and culture
Infected skin may be treated with topical application of antifungal agents miconazole,nystatin and clotrimazole
Refractory lesions oral griseofulvin and itraconazole, terbinafine
Infections of hair and nails usually require systemic ( oral) therapy

35. SUBCUTANEOUS MYCOSES( dermis, subc tissues and Bone)
Causative organisms reside in the soil and in decaying or live vegetation
Almost always acquired through traumatic lacerations or puncture wounds
Common among those who work with soil and vegetation and have little protective clothing
Not usually transmitted humans to humans
Usually confined to tropics and subtropics with exception of Sporotrichosis in USA

36. Sporotrichosis Sporothrix schenckii-dimorphic fungus
Granauloma ulcer at a puncture skin usually a thorn prick and may produce secondary lesions along draining lymphatics
In most disease is self-limiting may exist in chronic form
Treatment oral itraconazole
Chromomycosis : Phialophora or Cladosporium species

37. Mycetoma Madurella grisea, Actinomadura madura
Localized abscess usually on the feet, that discharge pus serum and blood
Has coloured grains( compact hyphae) black, white, red or yellow depending on organism

38. Aspergillus fumigatus Crytococcus neoformans
SYSTEMIC MYCOSES
Systemic infective agents
Blastomyces dermatitidis
Coccidioides immitis
Histoplasma capsulatum
Paracoccidioides brasiliensis
Absidia corymbifera
Aspergillus fumigatus
Candida albicans
Crytococcus neoformans
Pneumocystis carinii
Rhizomucor pusillus
Rhizopus oryzae (R.arrhizus)
Opportunistic fungal
Pathogens

39. Eastern US
Males
Diagram of Systemic mycoses(dimorphic, yeast in infective tissue)

40. Clinical significance
Simliar to Tuberculosis in that asymptomatic primary infection is seen whereas chronic pulmonary or disseminated infection rare
In the immunocompetent usually mild and self limiting
In the immunocompromised the same infections can be life threatening

41. Coccidiodomycosis Coccidioides immitis
Most in arid areas of south-western US
In the soil forms arthrospores
Spores airborne , germinate in the lungs and produce sphercules filled with many endospores- new spherule
In disseminated cases lesions in the bone or CNS -meningitis

42. Histoplasmosis Histoplasma capsulatum
In the soil conidia, germinate lungs into yeast-like cells
Becomes engulfed by macrophages and XX
Benign self-limiting or chronic, progressive , fatal
Disseminated disease only fungus intracellular RES parasitism
Area Ohio and Mississippi River area
DX: Culture or Exoantigen (immunodiffusion assay)
AIDS patients at particular risk
Treatment : Amphotericin
or Itraconazole

43. OPPORTUNISTIC PATHOGENS
Absidia corymbifera
Aspergillus fumigatus
Candida albicans
Crytococcus neoformans
Pneumocystis carinii
Rhizomucor pusillus
Rhizopus oryzae (R.arrhizus)

44. OPPORTUNISTIC MYCOSES
Those that affect the immunocompromised but are rare in normal individual
Organ transplantation, post chemotherapy for cancer, immunodeficient due to Aids and congenital immunodeficiency states
Candida species most commonly occurring fungal pathogen in the ICU setting

45. CANDIDIASIS(candidiosis)
Candida albicans and other candida species which are normal flora in the mouth, skin , vagina and intestines
May occur as a results of overgrowth as suppression of bacteria by antibiotics
Manifestations depend on the site e.g. oral candidiasis and vaginal candidiasis and disseminated candidiasis in cancer patients, post GI surgery and AB`s, systemic corticosteroids

46. Risk Factors for Candida Infection
Cellular Immunodeficiency
Antibiotic Use
Moisture area
Age
Hormonal Influence
General debility
Interference with Normal flora
Mechanical factors
Pregnancy
Oral Contraceptives
Diabetes mellitus
Administration of corticosteroids

47. Vulvovaginal candidiasis
Treatment miconazole, clotrimazole topically or oral fluconazole
Or itraconazole

48. Candida wet preparation

49. Candida species-Gram stain

50. Candida culture-24 hours

51. Mucosal Candidiasis
Pain, redness and sometimes a whitish coating or discharge of the mucosa
Oral candidiasis
Nappy rash candidiasis
Vaginal candidiasis
Esophageal Candidiasis
Chronic form

52. MUCOCUTANEOUS CANDIDIASIS
Cellular deficiency results in chronic mucocutaneous candidasis

53. Oral Candidiasis Occurs in infants without any predisposing factors
Usual predisposing factors
Seen in patients taking antibacterials
Pain, redness and sometimes a whitish coating or discharge of the mucosa
Candida present in small numbers on the mucosa and the problem arises when it overgrows

54. Eosophageal Candidiasis
Orophargneal candidiasis may progress to eosophageal candidiasis
Manifestataion of AIDS
Also occurs in those who have predisposing factors but are HIV-negative
Treatment: fluconazole,itraconazole, voriconazole or amphotericin

55. Vaginal Candidiasis May occur without any obvious predisposing factors
May occur frequently
Treatment:
Creams and ointments: Clotrimazole 1% , Miconazole 2%
Tablets/Pessaries: Clotrimazole, Miconazole, Terconazole, Nystatin
Oral Therapy: Fluconazole, Itraconazole

56. NAIL CANDIDIASIS
Paronychia
Oral therapy-fluconazole etc

57. DISSEMINATED CANDIDIASIS
Treatment amphotericin or fluconazole

58. Severe candida Infections
May cause candidaemia, opthalamitis, hepatosplenic candidiasis,
Line infections, secondary peritonitis and urinary tract infections in
Hospitalised patients
As well as mucosal candidiasis
Of Note: candida may contaminate sputum specimens

59. CRYTOCOCCOSIS Crytococcus neoformans, found worldwide
Especially found in soil containing bird(esp. pigeons) droppings
Characteristic thick capsule that surrounds budding yeast cell –seen Indian Ink
Most common form is mild subclinical lung infection
In the immunocompromised often disseminates to the brain , meningitis often fatal
However half those with crytococcal meningitis have no obvious immune deficiency
bird

60. CRYTOCOCCUS NEOFORMANS
In Aids patients it is the second most common fungal infection
after candida , potentially the most serious
Treatment: Amphotericin and flucytosine for meningitis and if AIDS
Subsequent suppression with fluconazole

61. ASPERGILLOSIS
Several species of genus Aspergillus, mostly Aspergillus fumigatus
Worldwide distribution, ubiquitous
Filamentous molds, produce large numbers of conidiospores
Reside in soil, decomposing organic matter and dust, associated outbreaks n hospitals with construction work
Disease presentation depends on immunologic status of patient

62. Disease caused by Aspergillus
Allergic Bronchopulmonary Aspergillosis
Farmer`s lung
Invasive Aspergillosis
Aspergilloma

63. Aspergillus fumigatus

64. Disease caused by Aspergillus
Allergic Bronchopulmonary Aspergillosis: in this condition the mould colonises the mucosal surface of lower respiratory tract but does not invade the mucosa. There is intense hypersensitivity response to the Aspergillus antigens> impairment of lung function. Associated abnormal findings on X-ray and asthma like symptoms

65. Farmer`s Lung
Syndrome of shortness of breath typically occuring several hours after exposure to mouldy hay. Antibodies (IgG not IgE) form a precipitate with aspergillus antigen in the alveolar walls and an inflammatory cascade is initiated

66. Allergic Aspergillosis
Relatively rare, can arise from inhalation of spores, without subsequent extensive spore germination hyphal invasion
The allergic reaction results in bronchial constriction
Diagnosis by immunoelectrophoresis

67. ASPERGILLOSIS Acute Aspergillus infections
Most severe and often fatal form of aspergillosis is acute invasive infection of the lungdissemination to brain etc
Less severe form gives rise to a fungus ball( aspergilloma) , a mass of hyphal tissue that forms in lung cavities derived from prior disease

68. ASPERGILLOMA
Diagnosis in the lab by staining and culture: characterisitic V-shaped
Hyphae, Septated and spore forming structures
Treatment Surgical removal of mass and amphotericin
Risk of massive haemoptysis

69. INVASIVE ASPERGILLOSIS INFECTION
Often treated empirically, using risk assessment and
CT(spiral) to assist in diagnosis
Treatment Amphotericin( or voriconazole) and supportive therapy
NEJMED 2002 Aug 8:347(6);408-15

70. MUCORMYCOSIS
Most often caused by Rhizopus oryzae and less often by other members of the Mucorales such as Absidia corymbifera, Rhizopus pus
Ubiquitous in nature, spores found in great abunance on rotting fruit and old bread
Usually restricted to those with underlying conditions such as burns, leukaemia or diabetus mellitus
The most common form of the disease can be fatal within a week-Rhino cerebral Mucormycosis

71. MUCOR MYCOSIS/RHIZOPUS

72. Rhinocerebral Mucormycosis
Infection begins in the nasal mucosa or sinuses and progresses to the
Orbits, the palate and the brain
Treatment: Surgical debridement of necrotic tissue , correction of
Underlying disorder and Amphotericin

73. RHIZOPUS from Skin Scrapings

74. PNEUMOCYSTIS CARINII PNEUMONIA (PCP)Now known as PNEUMOCYSTIS JIROVECI Frenkel 1999
Caused by a unicellular eukaryote, Pneumocystis carinii
Before the use of immunosuppressive agents and the onset of the AIDS epidemic , PCP was a rare disease
It is one of the most common opportunisitic diseases of individuals with HIV-1 and usually fatal if untreated
It does not contain ergosterol and is extremely difficult to culture (requires )cultured

75. PCP
Various cellular forms encysted group of dormant cells and vegetitive form –trophozoite
Ubiquitous
Activation of preexisting dormant cells in the lungs in immunodeficient persons
The encysted forms induce an inflammination of the alveoli-exudate which blocks gas exchange
Diagnosis by microscopic examination , by silver stain or fluorescence of bronchial washings or biopsy

76. Pneumocystis carinii in Alveoli
Treatment: Combination sulfamethoxazole and trimethoprim,
Pentamine and additional agents may also be used
Can be used prophylaxically to prevent infection

77. Pneumocystis carinii (jiroveci) pneumonia

78. LABORATORY IDENTIFICATION
Standard media –Sabouraud`s agar, potato dextrose agar, low ph 5.0 , inhibits bacterial growth but allows fungal colonies to form
Cultures can be started from spores or hyphae fragments
Specimens: blood, pus, CSF, sputum, tissue biopsies, skin scrapings , nail clippings
Identification by the morphology of conidia structures and carbonhydrate assimiliation tests

79. LABORATORY DIAGNOSIS OF FUNGAL INFECTION
Specimens
Depends on site of infection
Systemic: -Blood culture( really only useful for yeast-low sensitivity) or
- antigen testing e.g.crytococcal and histoplamsosis antigen
Pneumonia: Bronchoscopy washings or brushings for staining and fungal culture or bronchial biopsy

80. LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS
Meningitis: Cerebrospinal fluid for Lactophenol blue staining and indian ink and crytococcal antigen and fungal culture
If Skin infection require skin scrapings
If nail infection require nail clippings
Galactomannan antigen testing for aspergillus infection

81. LABORATORY DIAGNOSIS FUNGAL INFECTIONS
Types of tests carried out
Fungal Staining – Lactophenol blue staining or wet prep using KOH to dissolve tissue material or Calcofluor (fluorescence stain)
Fungal culture on media that encourages fungal growth e.g. PDA
Antigen Testing i.e. to test for antigen present in the wall of fungus e.g crytococcal antigen, galactomannan used in serum and CSF samples
Molecular Methods not used on a routine basis on samples(as yet)

82. MANAGEMENT OF FUNGAL INFECTIONS
Some such as superfical skin infections require topical therapy only with cream e.g.miconazole cream
Some require local therpy e.g. pessaries for vaginal candidasis
Some require oral therapy for skin and nail infections up to 1 year e.g. terbinafine
In the immunocompromised systemic therapy required e.g. fluconazole i./v or amphotericin, voriconazole

83. MANAGEMENT OF FUNGAL INFECTIONS
Important to diagnose fungal infections early in the immunocompromised as there is a high mortality associated with infection
Empirical therapy often started in advance of laboratory diagnosis in these patients

84. Antifungal Agents: Families
Azoles
Allylamines
Benzofurans
Polyenes Macrolides
Pyrimidines
Lipopeptides
Imidazoles Triazoles
Ref: Antifungal Drug Resistance. Clinical Infectious Diseases. 2003:36
(Suppl 1) s31-41.

86. Azoles
Azoles
Causes Inhibition of C-lansterol 14 α demethylase, (an enzyme required for the synthesis of ergosterol) by binding to cytochrome P450
Resistance may be intrinsic or acquired
Imidazoles Triazoles
Voriconazole

87. Allyamines
Inhibits squalene epoxidase, an enzyme essential for synthesis of ergosterol
Drug acculmulates in nails, skin and fat
Very useful for nail infections

88. Polyene Macrolides Amphotericin, nystatin
Antifungal activity by binding to membrane sterols such as ergosterol and they increase membrane permeability and leads to cell death
Higher concentrations inhibits Chitin synthase
Active against Aspergillus spp, Candida species ,Crytococccus neoformans , Zygomycetes etc

89. Amphotericin Numerous forms
Pastilles, Parenteral forms: amphotericin B, deoxycholate form, colloidal form, Liposomal form
Toxicity: Dose dependent reduction in GFR, by direct vasoconstritive effect on afferent renal arterioles, destruction of renal tubular cells and basement membrane and loss of functioning units
Also nausea .vomiting, phlebitis and ACUTE REACTION: fever,chills,tachyapnea

90. Pyrimidines Fluorine analogue of a normal cell constituent cytosine
Demination results in 5-fluorouracil, to 5-flurodeoxyuridylic acid monophosphate, a non-competitive inhibitor of thymidylate synthetase
Used particularly in crytococcal meningitis-74% of serum levels

91. Benzofurans Griseofulvin
Inhibits nucleic acid synthesis, macrotubule formation and chitin formation
Active against ringworm, not candidia or tinea versicolor

92. Lipopeptides Echinocandins, derivatives of pneumocandin BO
Inhibition of 1,3-ß- glucans in the fungal wall, that is glucan synthase inhibitor
Active candida, aspergillosis and pneumocystis carinii in vitro
Licensed for refractory candida( esophageal) infections and invasive Aspergilllosis