Mycology Introduction, Systemic Fungal Infections, And Opportunistic Fungal Infections

General Fungal Characteristics

• Eukaryotic

  • nucleus, nuclear membrane, mitochondria

• Sterols in the cell wall

  • lipids, play role in membrane integrity

• Chemoheterotrophs

  • require organic nutrition

  • aerobes, grow best at neutral pH

• Saprophytic: living on dead or decaying organisms

• acquire food by absorption

• produce sexual and asexual spores (conidia)

  • most produce asexually

Most fungi exist as:

• Molds: multicellular, filamentous form of fungi consisting of thread-like filaments to form fuzzy colonies

• Yeasts: unicellular, produce circular, restricted, pasty, or mucoid colonies

Fungi can also be:

• dimorphic: exhibit a yeast or mold phase dependent on temperature

  • if ingested, would be a yeast due to higher body temps

Filamentous Fungi (mold)

• composed of microscopic filaments called hyphae that branch to form a network called the mycelium (colony)

  • extend over or through whatever substrate the fungus is using as a food source

  • Aeriel: hold reproductive structures (spores, seeds)

  • Vegetative: absorb nutrients

• Hyphae

  • Shape: antler, racquet, spiral, rhizoid

  • Septate: perpendicular cross-walls

    • can be sparse or common

  • pigmentation

    • Hyaline (non pigmented hyphae)

    • Dematiaceous (dark pigmented due to presence of melanin in cell wall)

• Fungal Spores (conidia)

  • Functions:

    • means of dispersal

    • means of survival (low metabolic state of activity)

    • Dormant state; will germinate when environment conditions are favorable again for growth

  • vary in size, shape, and color

  • unicellular or multicellular

Unicellular Yeast

• Capable of reproducing asexually and sexually

  • Asexual: budding or binary fission (most bud)

Candida Cell Wall

• mannoproteins for cell stability

• Chitin and Glucan (polysaccharides) for structural integrity

Cryptococcus Cell Wall (yeast)

• Capsule (polysaccharide)

  • separates yeast species

  • masks antigens

  • virulence factor (protection against and interfere with host immune cells)

• Chitin and Glucan (polysaccharides) for structural integrity

• Melanin: virulence factor

Aspergillus Cell Wall (mold)

• Galactomannan (polysaccharide): released during tissue invasion

  • can be tested for in a clinical setting for diagnosis

• Chitin and Glucan (polysaccharides) for structural integrity

• Rodlet (protein) virulence factor

Fungal Diseases

• Grouping is traditionally based on botanic taxonomy (classification)

  • Clinical microbiology groupings based on mycoses (fungal diseases)

• Superficial: confined to the outermost dead layer of the skin or hair

• Cutaneous: affects the keratinized layer of the skin, hair, or nails

• Subcutaneous: deeper skin layers including muscle, bone and connective tissue without dissemination to distant sites

• Systemic: affects the internal organs or deep tissues of the body

• Opportunistic: found primarily in immunocompromised persons; infections of a great variety of tissues

  • pose a significant diagnostic challenge due to complexity due to complexity of the patient population at risk and increasing array of fungi that can infect immunocompromised individuals

Innate Resistance

• despite constant exposure to the infectious forms of various fungi (between 1000 to 10 BILLION spores daily from vast number of species)

• The key factor that provides barrier is out internal temp — fungi unable to adapt to higher temps

• A healthy immunocompetent individual will have high innate resistance to fungal infections

Mode of Transmission

• Dependent on type of mycoses (infections)

  • Superficial & Cutaneous: person-to-person contact, person-to-animal contact, as well as fomites (objects/materials that carry infectious agents)

  • Subcutaneous: through the skin, after a cut or other trauma to the skin

  • Deep mycoses: opportunistic growth in immunocompromised; inhalation of spores/conidia; or presence of intravenous device

    • most common mode

Lab Values to Support Fungal Infection

• increased WBC count

• increased lymphocytes and monocytes

• mod-marked increase in total protein

• normal-to-low glucose

• lactate > 25mg/dl

Specimen Collection, Transport, Handling, And Growing

• Based on information from clinical examination and radiographic studies and consideration of the most likely fungal pathogens that may cause a specific type of infection

• Collected aseptically or after proper cleaning/decontamination of the site sampled

  • adequate amount of clinical material is collected in a sterile, leak proof container accompanied by a relevant clinical history, to be tested immediately or stored at 4^oC

• Specimens processed under a bio safety cabinet

• Identification methods:

  • direct microscopy, culture, biochemicals and susceptibility testing

  • automated identification systems

  • immunologic methods

  • molecular methods

• Culture Time and Conditions

  • Time: growth of yeast is usually detected within 48-72 hours, mold could take up to 4-6 weeks

  • Temp: fungal cultures grown at 25-30^oC, while dimorphic fungi are grown at mold temps and yeast temps for conversion to yeast phase which could take 7-14 days

• Culture Media

  • Enriched media to ensure growth of fastidious thermally dimorphic fungi (blood enrichment)

  • General purpose media to ensure growth of a varieties of molds and yeasts

    • contain no antimicrobial agents

    • contain an antibacterial

    • contain an antibacterial and antifungal (cycloheximide)

      • cycloheximide inhibits Cryptococcus neoformans, Mucorales, Candida spp., Aspergillus spp., Histoplasma spp.

Colony Morphology

• Texture: height of aerial hyphae

  • wooly/cottony (dense, high)

  • velvety (low aerial hyphae)

  • granular (flat, dense aerial hyphae)

• Color: surface and reverse

• Topography: designs of hills and valleys

Microscopy

Stains & Reagents

• Lactophenol cotton blue:

  • kills any organism and lactic acid acts as a clearing agent and preserves fungal structures

  • stains the chitin in fungal cell walls

• Potassium Hydroxide

  • 10-20% solution of KOH dissolves keratin

    • contrast black / white staining gives outline of organism

• Calcofluor White

  • fluorochrome stain that binds to cellulose and chitin in fungal cell walls

  • appears fluorescent green

• Gram Stain

  • fungi stain gram positive (purple)

Identification of fungi by observance of asexual structures (sporangiospores or conidia)

Microscopy Conidia

Antifungal Agents

• Agents can have broad or narrow spectrum

• Fungistatic (inhibits and slows growth) or Fungicidal (kill organism)

• Systemically active and topical Agents

Polyenes

• systemically active agent

• Amphotericin B and Nystatin

• Work by binding to ergosterol, the principal membrane sterol of fungi. Binding produces ion channels, which destroy the osmotic integrity of the fungal cell membrane and lead to leakage —> eventually death

• also bind to cholesterol, main membrane sterol of mammal cells (makes polyenes toxic)

  • nephrotoxicity (kidney damage)

• Broad spectrum of activity

  • dimorphic fungi

  • opportunistic fungi

  • Candida spp.

  • Cryptococcus neoformans

• Fungicidal

Azoles

• Systemically active agent

• Inhibit fungal Cytochrome P-450-dependent enzyme, lanosterol 14-alpha-demethylase

  • enzyme is involved in the conversion of lanosterol to ergosterol

  • inhibition disrupts cell membrane synthesis

• fungistatic in yeast and fungicidal in mold

Echinocandins

• systemically active agent

• Inhibit synthesis of 1,3 beta-glucans, constituents of fungal cell wall

• Fungicidal in Candida and fungistatic in Aspergillus

• low toxicity - we don’t have cell walls

• Ex. Caspofungin

Flucytosine

• systemically active

• interferes with the synthesis of DNA, RNA and proteins of the fungal cell wall

• Enters the cell wall via cytosine permease; converted by cytosine deaminase in fungal cells to fluorouracil — which competes with uracil and results in RNA miscoding

• fluorouracil is metabolized to 5-fluorodeoxyuridylic acid which halts DNA synthesis

• Fungistatic

• limited spectrum of activity

• typically used in combination with another antifungal agent due to resistance

Allylamines

• Systemically active

• Ex. Terbinafine

• Inhibit the enzyme squalene epoxidase which results in a decrease in ergosterol and an increase in squalene, producing a toxic effect within the cell membrane

• broad spectrum of activity (dermatophytes, yeasts, molds)

• Fungicidal

Topical Active Agents

• Available in most classes of antifungal agents

• creams, lotions ointments, powders, and spays

• treatment of superficial, cutaneous, and mucosal infections

• Use of topical vs systemic therapy is dependant on status of host as well as type and extent of infection

Antifungal Resistance

• Resistance develops slowly and involves the emergence of intrinsically resistant species or a gradual, stepwise alteration of cellular structures or functions

• Mechanisms

  • biofilm formations — reduced penetration of antifungals

  • Efflux pumps — reduces accumulation of drug

  • Target alterations/mutations

  • overexpression of target

Pathogenesis of Fungal Disease

• few fungi are virulent enough to be considered primary systemic pathogens or capable of initiating infection in an immunocompetent person

• Most are considered opportunistic pathogens, only causing infections when there are disruptions to protective barriers (skin) or when there is defects in the immune system

Primary Systemic Pathogens

• agents of respiratory infection

• have a saprobic phase (found on soil or decaying vegetation) thats forms airborne cells

• And a parasitic phase (adapted to growth at 37^oC and reporduces in host

Blastomyces dermatitidis

• geographic distribution: North America (OH and MS river valleys) Africa, and SW Asia

• Ecologic niche: soil, woody plants and decaying matter

• 250 cases/yr

• Dog infection rate is 10x that of humans

• Route of infection: inhalation of conidia, inoculation od soil, dog bite

• Clinical Syndromes: severity is dpendent on extent of exposure and immune status

• Symptoms occur in fewer than half of infected individuals

  • incubation 1 to 15 weeks

• Illness: pulmonary disease or extrapulmonary disseminated disease (heart)

  • pulmonary blastomycosis is asymptomatic or presents as mild-flu like illness

    • ~50% are asymptomatic

  • classic form of blastomycosis: chromic cutaneous mycosis

• Pathogenicity:

  • following inhalation, conidia (2-10um) convert to yeast (8-30um)

    • larger yeast resist immune attack

  • localized yeast invasion of host invokes an inflammatory response

  • yeast escape recognition by macrophages (shed antigen from cell surface and modify cell wall)

• Diagnosis

  • microscop detection in tissue

  • broad-base budding yeast

  • serologic assays not useful

  • antigen test

Coccidioides immitis / posadasii

• Geographic distribution: Southwestern US, Mexico, Central & South Americas

  • C. immitis is localized to California

  • C. posadasii accounts for majority of infections outside of CA

• Ecological niche: soil, dust, bat and bird droppings — nitrogenous (alkaline)

• Cycles of heavy rain allows growth, drought and wind allows dispersal

• Route of infection: inhalation of arthroconidia

  • only a few can produce primary coccidiomycosis

• Clinical syndromes: can be either pulmonary or extrapulmonary

• asymptomatic in 30-60% of patients

• incubate for 1-3 weeks

• last for a few weeks to months

• Symptoms:

  • fever, cough, headache, muscle aches and joint pain

    • valley fever

  • cutaneous manifestations (rash)

  • chronic pneumonia

  • meningitis

  • bone and joint infection

• diagnosis:

  • microscopic detection of the fungus in tissue or other clinical material with confirmation by culture

    • spherules containing endospores in tissue

  • serologic testing (IgM and IgG)

  • antigen test

Histoplasma capsulatum

• Distribution: North America (OH and MS river valleys, Mexico) Central & South America, Africa, Asia, Australia

• Ecological niche: soil with high nitrogen, bat and bird droppings

• ~90% infected are male

• Route of Infection: inhalation of microconidia

• Clinical syndromes:

  • severity depends on extent of exposure and immune status

  • Symptoms: like pneumonia occur in ~10% of infected individuals

  • incubation: 4-18 days

  • in the event of heavy inoculum, pulmonary histoplasmosis occurs

    • 1 in 2000 adults

  • reactivation common among immunocompromised

  • an estimated 60-90% of people living around Ohio and Miss. River valleys have been exposed

  • incidence higher in ages 65+

• Diagnosis:

  • microscopic detection in tissue

    • small, oval narrow budding yeast

    • serologic testing in blood or urine

    • antigen test

Talaromyces marneffei

• Distribution: southcentral, southeast and east Asia

• Ecologic niche: soil and has been isolated from bamboo rats

• Route of infection:

  • inhalation of conidia

  • traumatic implantation

    • eating bamboo rat

• Clinical syndromes:

  • disseminated skin infections (skin, soft tissue)

  • occurs primarily in immunocompromised people in Thailand and SE China (early indicator of HIV)

  • resembles histoplasmosis, cryptococcosis and tuberculosis

• Diagnosis:

  • microscopic detection in tissue

  • morphology: elliptical fission yeast that are intracellular

  • no serological testing but under development

  • molecular assay

Systemic Fungi treatment options

• clinical form, severity, and immune status, antifungal toxicity must be taken into consideration

• treatment duration may range from 3 months to year(s) AIDS patients require lifelong treatment

Oppritunistic Mycoses

• Pose significant diagnostic challenge:

  • complexity of the patient population (immunocompromised) and increasing array of fungi that can infect these individuals

Candidiasis (yeast)

• more than 100 species, only ~20 have have been implicated in clinical infections

  • C. albicans accounts for 90-100% of mucosal isolates and 50-70% of bloodstream infections

  • C. glabrata

  • C. parapsilosis

  • C. tropicalis

• Emerging pathogen: C. auris

  • causes invasive infection

  • Japan, 2009

  • Often resistant to multiple antifungals

  • challenging to identify

  • health-care associated transmission

• Epidemiology:

  • known colonizers of humans an warm-blooded animals

  • Primary site: GI tract from rectum to mouth

  • commensals in the vagina and urethra, on the skin, under nails

  • most represent endogenous infections

    • exogenous infections- can transmit person to person

• Clinical syndromes: cause infection of virtually every organ system

  • range from superficial mucosal and cutaneous to widespread hematogenous dissemination involving target options

    • liver, kidney, heart, brain and spleen

• Candidiasis

  • diagnosis: collection of tissue or fluid followed by direct microscopy

  • Treatment, prevention, and control:

    • generally treated with single antifungal agent

    • remove source of infection and enhance immune system

    • remove catheters, drain absesses

Cryptococcus neoformans

• distributed worldwide

• ecological niche: soil contaminated with bird droppings

• Route of infection: inhaling aerosol cells

• clinical syndromes:

  • can cause latent or systematic disease (basec on virulence and immune status)

  • can cause disease in healthy individual, more frequent and severe in immunocompromised

    • incubation of 6-8 weeks

  • presents as either pneumonia or a central nervous system infection secondary to hematogenous and lymphatic spread

  • meningitis in 40-80% of cases

• clinical syndrome: disseminated disease includes skin lesions and ocular infections

• diagnosis:

  • microscopy detection of yeast and examination of CSF might reveal characteristic encapsulated budding yeast

  • direct detection of capsular polysaccharide antigen

• treatment:

  • cryptococcal meningitis and other disseminated forms are fatal if left untreated

  • severe infection: amphotericin B + flucytosine for 2 weeks followed by 8 weeks of fluconazole

  • mild to moderate: fluconazole

  • AIDS patients require lifelong treatment

Cryptococcus gattii

• located in pacific northwest and moved down to southeast US

  • important to be distinguished from C. neoformans infections bc C. gatti requires lengthier treatments and more aggressive

• typically infections patients without HIV

• most infections stay pulmonary

• ecological niche: woody materials

Aspergillosis

• distributed worldwide

• ubiquitous in air, soil, decaying matter

  • A. fumigatus

  • A. flavus

    • common in hospital environments

• at-risk populations: “patients with”

  • prolonged neutropenia

  • immunodeficient or immunocompromised

  • corticosteroids

• Aspergillosis

  • route of infection: inhalation of conidia

  • incubation of 3 days —> 3 weeks

  • allergic bronchopulmonary aspergillosis (ABPA): manifestations based on hypersensitivity to antigens, causes wheezing and coughing

  • sinusitis

  • invasive aspergillosis: 70% mortality! seen in neutropenic and immunodeficient patients and cystic fibrosis patients

• clinical syndromes:

  • produce aflatoxin when it A. flavus contaminates food

    • peanuts, dried fruits, corn, rice

  • symptoms: nausea, vomiting, abdominal pain, convulsions

  • chronic exposure associated with liver cancer

  • safe levels regulated by FDA

• Diagnosis:

  • aspergillus galactomannan antigen in serum

  • isolation from blood is rare

  • specimen source: bronchoalveolar valve

• Treatment:

  • prevention in high risk patients

  • reconstitute host defenses

  • surgical removal

  • antifungal therapy — amphotericin B

Microsporidia

• spore forming

• 15 species are pathogens to humans

• distrubuted worldwide and have a wide host range

• 7-50% of aids patients infected

• transmitted by inhalation of spores

• unknown incubation period

• Clinical syndromes:

  • double infections with Cryptosporidium are common

  • symptoms: profuse, watery diarrhea

    • dissemination to involve loss of vision, neurologic disturbances, hepatitis, UTI, pulmonary infections

    • clinical manifestations are very diverse, varying to causal species

• Diagnosis:

  • detection of organisms in biopsy and examination of cerebrospinal fluid and urine

  • molecular methods

• treatment and prevention:

  • restoration of immune system

  • improved sanitation

Mucormycosis

• distributed worldwide

• common bread mold, also found in soil and decaying vegetation

• common pathogens:

  • Rhizopus

  • Mucor

  • Lichtheimia

• clinical syndromes:

  • 1.7 million annual US infections

  • infections with mortality ranging from 70-100%

  • infections acquired by inhalation, ingestions, or contamination of wound from sporangiospores

• clinical syndromes

  • gastrointestinal mucormycosis

    • more common in young children

  • disseminated mucormycosis

    • fungus spreads into bloodstream to affect various parts of body

    • affects the brain and heart

  • diagnosis:

    • tissue for direct microscopy and culture

    • specimens from nasal scrapings, sinus aspirates, biopsy material

    • serology not useful

    • negative culture results common even when hyphae are seen in tissue

  • Treatment:

    • amphotericin B often supplemented with surgical debridement

    • duration of treatment is individualized

Hyalohyphomycosis

• 100 known organisms

• infections caused by non pigmented mold

• distributed worldwide

• commonly encountered in lab environment as saprobes

• many infections acquired during construction/demolition and hospital acquired

• mostly seen in immunocompromised

• Fusarium

  • causes mycotic keratitis

  • disseminated infection in immunocompromised

• diagnosis

  • fungi can be recovered from patients having no evidence of infection

  • culture for identification

• Treatment:

  • resistance to antifungals

  • immune reconstitution

  • surgical removal

Phaeohyphomycosis

• 100 known organisms

• caused by pigmented mold

• distributed worldwide

• everywhere in nature

• clinical syndromes:

  • disseminated infections or localized infections of the lung, paranasal sinuses and CNS

  • mostly seen in immunocompromised

• diagnoses:

  • culture for identification

  • fungi can be recovered from patients having no evidence of infection

• treatment:

  • amphotericin B

  • surgical removal

Pneumocystis

• P. jiroveci causes illness almost exclusively in immunocompromised patients especially with HIV

• prefer humans and animals

• transmit by inhalation or person-person

• clinical syndromes:

  • pneumonia

  • extrapulmonary manifestations

    • lymph nodes, spleen, bone marrow, liver, eyes, ears

• diagnoses:

  • microscopic examination of clinical material

  • molecular assay

• Treatment:

  • SXT for 3 weeks and prophylaxis

    • interferes w acid synthesis