MEDICALLY IMPORTANT FUNGI and ANTIFUNGAL THERAPY DR. BREIDA BOYLE

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

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

PATHOGENIC FUNGI TRUE PATHOGENS OPPORTUNISTIC PATHOGENS

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

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

PATHOGENIC FUNGI TRUE PATHOGENS OPPORTUNISTIC PATHOGENS

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

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

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

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

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

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

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

REPRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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 10-14 days for growth Macroscopic and microscopic identification of colonies

Fungal elements/hyphae

T.mentagrophytes

T.mentagrophytes

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Candida wet preparation

Candida species-Gram stain

Candida culture-24 hours

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

MUCOCUTANEOUS CANDIDIASIS Cellular deficiency results in chronic mucocutaneous candidasis

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

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

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

NAIL CANDIDIASIS Paronychia Oral therapy-fluconazole etc

DISSEMINATED CANDIDIASIS Treatment amphotericin or fluconazole

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

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

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

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

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

Aspergillus fumigatus

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

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

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

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

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

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

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

MUCOR MYCOSIS/RHIZOPUS

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

RHIZOPUS from Skin Scrapings

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

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

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

Pneumocystis carinii (jiroveci) pneumonia

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

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

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

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)

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

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

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.

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

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

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

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

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

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

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