MEDICALLY IMPORTANT MEDICALLY IMPORTANT FUNGI and ANTIFUNGAL FUNGI and ANTIFUNGAL

THERAPYTHERAPY

DR. BREIDA BOYLE

INTRODUCTIONINTRODUCTION

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

INTRODUCTIONINTRODUCTION

MYCOSESMUCOSAL: limited to mucosaeCUTANEOUS: limited to the dermisSUBCUTANEOUS : when infection

penetrates significantly beneath the skinSYSTEMIC : when the infection is deep

within the body or disseminated to internal organs

PATHOGENIC FUNGIPATHOGENIC FUNGI

TRUE PATHOGENS

OPPORTUNISTICPATHOGENS

TRUE PATHOGENSTRUE PATHOGENS

Epidermophyton speciesMicrosporum speciesTrichophyton species

Actinomadura maduraeCladosporium

Madurella griseaPhialophora

Sporothrix schenckii

Blastomyces dermatitidis Coccidioides immitis

Histoplasma capsulatumParacoccidioides brasiliensis

Cutaneous infective agents Subcutaneous infective agents

Systemic infective agents

OPPORTUNISTIC OPPORTUNISTIC PATHOGENSPATHOGENS

Absidia corymbiferaAspergillus fumigatus

Candida albicansCrytococcus neoformans

Pneumocystis cariniiRhizomucor pusillus

Rhizopus oryzae (R.arrhizus)

PATHOGENIC FUNGIPATHOGENIC FUNGI

TRUE PATHOGENS

OPPORTUNISTICPATHOGENS

CLASSIFICATION OF FUNGICLASSIFICATION OF FUNGI

Depends on :Characteristic StructuresHabitatsModes of Growth Modes of ReproductionClinical SettingDNA Homology

Cell Wall and MembraneCell 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 ergosterolpores that disrupts membrane function cell death

Cell MembraneCell 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

HABITATHABITAT

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 MODES OF FUNGAL GROWTHGROWTH

FILAMENTOUSMOLDS

UNICELLULARYEASTS

However there are some dimorphic fungi ( they switch between these Two forms depending on their environment)

Filamentous (mold-like) FungiFilamentous (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 FUNGIYEAST 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

REPRODUCTIONREPRODUCTION

SPORULATIONSPORULATION

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 SPORULATIONASEXUAL SPORULATION

Colour of a particular fungus seen on bread, culture plate is due to theConidia, easly airborne and disseminated

(MITOSIS)

SEXUAL SPORULATIONSEXUAL SPORULATIONmeiosis

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

CUTANEOUS MYCOSESCUTANEOUS MYCOSES-DERMATOPHYTOSES-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

PATHOLOGYPATHOLOGY

Dermatophytes use keratin as a source of nutrition

Therefore they infect skin, hair, nailsAll 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 SIGNIFICANCECLINICAL SIGNIFICANCE

DERMATOPHYTOSESCharacterized 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)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)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)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/UNGUIUMTINEA 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 vesicolorTinea vesicolor

Pityrasis vesicolorDue to Malassezia furfur or Pityosporium

orbiculareTreatment , ketoconazole, fluconazole ,

itraconazole

Diagnosis of Dermatophyte Diagnosis of Dermatophyte InfectionInfection

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/hyphaeFungal elements/hyphae

T.mentagrophytesT.mentagrophytes

T.mentagrophytesT.mentagrophytes

TreatmentTreatment

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 SUBCUTANEOUS MYCOSES( dermis, subc MYCOSES( dermis, subc

tissues and Bone)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

SporotrichosisSporotrichosis 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

MycetomaMycetoma

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

SYSTEMIC MYCOSESSYSTEMIC MYCOSES

Blastomyces dermatitidis Coccidioides immitis

Histoplasma capsulatumParacoccidioides brasiliensis

Systemic infective agents

Absidia corymbiferaAspergillus fumigatus

Candida albicansCrytococcus neoformans

Pneumocystis cariniiRhizomucor pusillus

Rhizopus oryzae (R.arrhizus)

Opportunistic fungal Pathogens

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

Eastern US

Males

Clinical significanceClinical 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

CoccidiodomycosisCoccidiodomycosis

Coccidioides immitisMost in arid areas of south-western USIn the soil forms arthrosporesSpores airborne , germinate in the lungs and

produce sphercules filled with many endospores- new spherule

In disseminated cases lesions in the bone or CNS -meningitis

HistoplasmosisHistoplasmosis 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 riskTreatment : Amphotericin or Itraconazole

OPPORTUNISTIC OPPORTUNISTIC PATHOGENSPATHOGENS

Absidia corymbiferaAspergillus fumigatus

Candida albicansCrytococcus neoformans

Pneumocystis cariniiRhizomucor pusillus

Rhizopus oryzae (R.arrhizus)

OPPORTUNISTIC MYCOSESOPPORTUNISTIC 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)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 Risk Factors for Candida InfectionInfection

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 candidiasisVulvovaginal candidiasis

Treatment miconazole, clotrimazole topically or oral fluconazoleOr itraconazole

Candida wet preparationCandida wet preparation

Candida species-Gram stainCandida species-Gram stain

Candida culture-24 hoursCandida culture-24 hours

Mucosal CandidiasisMucosal Candidiasis

Pain, redness and sometimes a whitish coating or discharge of the mucosa

Oral candidiasisNappy rash candidiasisVaginal candidiasisEsophageal CandidiasisChronic form

MUCOCUTANEOUS MUCOCUTANEOUS CANDIDIASISCANDIDIASIS

Cellular deficiency results in chronic mucocutaneous candidasis

Oral CandidiasisOral 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 CandidiasisEosophageal Candidiasis

Orophargneal candidiasis may progress to eosophageal candidiasis

Manifestataion of AIDSAlso occurs in those who have predisposing

factors but are HIV-negativeTreatment: fluconazole,itraconazole,

voriconazole or amphotericin

Vaginal CandidiasisVaginal 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 CANDIDIASISNAIL CANDIDIASIS

Oral therapy-fluconazole etc

Paronychia

DISSEMINATED DISSEMINATED CANDIDIASISCANDIDIASIS

Treatment amphotericin or fluconazole

Severe candida InfectionsSevere 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

CRYTOCOCCOSISCRYTOCOCCOSIS

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

CRYTOCOCCUS CRYTOCOCCUS NEOFORMANSNEOFORMANS

In Aids patients it is the second most common fungal infection after candida , potentially the most seriousTreatment: Amphotericin and flucytosine for meningitis and if AIDSSubsequent suppression with fluconazole

ASPERGILLOSISASPERGILLOSIS

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 Disease caused by AspergillusAspergillus

Allergic Bronchopulmonary AspergillosisFarmer`s lungInvasive AspergillosisAspergilloma

Aspergillus fumigatusAspergillus fumigatus

Disease caused by Disease caused by AspergillusAspergillus

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 LungFarmer`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 AspergillosisAllergic 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

ASPERGILLOSISASPERGILLOSIS

Acute Aspergillus infectionsMost severe and often fatal form of

aspergillosis is acute invasive infection of the lungdissemination 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

ASPERGILLOMAASPERGILLOMA

Treatment Surgical removal of mass and amphotericinRisk of massive haemoptysis

Diagnosis in the lab by staining and culture: characterisitic V-shaped Hyphae, Septated and spore forming structures

INVASIVE ASPERGILLOSIS INVASIVE ASPERGILLOSIS INFECTIONINFECTION

Treatment Amphotericin( or voriconazole) and supportive therapy NEJMED 2002 Aug 8:347(6);408-15

Often treated empirically, using risk assessment and CT(spiral) to assist in diagnosis

MUCORMYCOSISMUCORMYCOSIS

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 MUCOR MYCOSIS/RHIZOPUSMYCOSIS/RHIZOPUS

Rhinocerebral MucormycosisRhinocerebral Mucormycosis

Infection begins in the nasal mucosa or sinuses and progresses to the Orbits, the palate and the brainTreatment: Surgical debridement of necrotic tissue , correction of Underlying disorder and Amphotericin

RHIZOPUS from Skin RHIZOPUS from Skin ScrapingsScrapings

PNEUMOCYSTIS CARINIIPNEUMOCYSTIS CARINIIPNEUMONIA (PCP)Now PNEUMONIA (PCP)Now

known as PNEUMOCYSTIS known as PNEUMOCYSTIS JIROVECI Frenkel 1999JIROVECI 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

PCPPCP

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 AlveoliPneumocystis carinii in Alveoli

Treatment: Combination sulfamethoxazole and trimethoprim, Pentamine and additional agents may also be usedCan be used prophylaxically to prevent infection

Pneumocystis carinii Pneumocystis carinii (jiroveci) pneumonia(jiroveci) pneumonia

LABORATORY LABORATORY IDENTIFICATIONIDENTIFICATION

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 LABORATORY DIAGNOSIS OF FUNGAL INFECTIONOF 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 LABORATORY DIAGNOSIS OF FUNGAL INFECTIONSOF FUNGAL INFECTIONS

Meningitis: Cerebrospinal fluid for Lactophenol blue staining and indian ink and crytococcal antigen and fungal culture

If Skin infection require skin scrapingsIf nail infection require nail clippings Galactomannan antigen testing for

aspergillus infection

LABORATORY DIAGNOSIS LABORATORY DIAGNOSIS FUNGAL INFECTIONSFUNGAL 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 MANAGEMENT OF FUNGAL INFECTIONSINFECTIONS

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 MANAGEMENT OF FUNGAL INFECTIONSINFECTIONS

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: FamiliesAntifungal Agents: Families

Azoles

Allylamines

Benzofurans

Polyenes Macrolides

Pyrimidines

Lipopeptides

Imidazoles Triazoles

Ref: Antifungal Drug Resistance. Clinical Infectious Diseases. 2003:36(Suppl 1) s31-41.

AzolesAzoles

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

AllyaminesAllyamines

Inhibits squalene epoxidase, an enzyme essential for synthesis of ergosterol

Drug acculmulates in nails, skin and fat

Very useful for nail infections

Polyene MacrolidesPolyene 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

AmphotericinAmphotericin

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

PyrimidinesPyrimidines

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

BenzofuransBenzofurans

GriseofulvinInhibits nucleic acid synthesis, macrotubule

formation and chitin formationActive against ringworm, not candidia or

tinea versicolor

LipopeptidesLipopeptides

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