Mycology 4-5

Inhalation of Spores

Direct Contact

Inoculation through Trauma

Endogenous Origin

Iatrogenic

Modes of Fungal Transmission and Infection

Inhalation of spores

– The most common route, especially for airborne fungi like Aspergillus, Histoplasma, and Coccidioides. Spores or conidia are inhaled and reach the lungs, initiating respiratory infection.

Direct contact

– Common in cutaneous and mucocutaneous mycoses such as Candida, Dermatophytes, and Sporothrix schenckii. Transmission may occur via skin trauma, contact with infected individuals, or fomites

Inoculation through Trauma

– Fungi such as Sporothrix and Fusarium may enter through puncture wounds or abrasion

Endogenous Origin

– Opportunistic yeasts like Candida albicans are part of normal microbiota and can cause infection when host defenses are compromised.

Latrogenic

– Infections can result from medical interventions such as catheterization, surgery, or use of contaminated medical devices.

Thermotolerance

Dimorphism

Capsule formation

Melanin production

Hydrolytic enzymes

Adhesins

Biofilm formation

Virulence Factors of Pathogenic Fungi

Thermotolerance

Ability to grow at 37°C is essential for survival in the human host.

Dimorphism

Some fungi, such as Histoplasma and Blastomyces, switch from mold to yeast form in host tissues, which enhances pathogenicity.

Capsule formation

– Cryptococcus neoformans produces a polysaccharide capsule that resists phagocytosis and suppresses immune responses.

Melanin production

– Pigment in fungi like Cryptococcus and Aspergillus protects against oxidative stress and antifungal agents.

Hydrolytic enzymes

– Fungi produce proteases, phospholipases, lipases, and elastases that degrade host tissues and aid in invasion.

Adhesins

– Surface molecules (e.g., Candida ALS proteins) facilitate binding to host cells.

Biofilm production

– Seen in Candida and Aspergillus species; enhances resistance to antifungal drugs and immune clearance

Adherence to Host Surfaces

Invasion of Host Tissues

Tissue Damage

Mechanisms of Fungal Adherence, Invasion, and Tissue Damage

Adherence to Host Surfaces

– Fungi utilize adhesins to bind to epithelial or endothelial cells, promoting colonization

Superficial invasion

Deep invasion

2 types of invasion of host tissues

Direct destruction

Indirect damage

2 types of tissue damage

Direct destruction

via enzyme secretion and metabolic by-products (e.g., reactive oxygen species).

Indirect damage

through host inflammatory response, which can exacerbate tissue injury.

Innate Immune Response

Adaptive Immune Response

Host Immune Response to Fungal Infections

Physical Barriers

Phagocytic cells

Pattern Recogniion Receptors (PRRs)

Innate immune response components

Neutrophils and Macrophages

are critical for killing fungi via phagocytosis and oxidative burst.

Dendritic cells

help in antigen presentation and triggering adaptive immunity.

Pattern Recognition Receptors (PRRs)

Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) such as Dectin-1 recognize fungal components like β-glucans and mannans.

Cell Mediated Immunity (CMI)

Humoral Immunity

Components of Adaptve Immune Response

Th1 responses

Th17 responses

Cell Mediated Immunity

Th1 responses

promote macrophage activation and fungal clearance via IFN-γ.

Th17 responses

enhance neutrophil recruitment and mucosal defense.

Humoral Immunity

Antibodies may neutralize fungi or opsonize them for phagocytosis, though less central than CMI

Antigenic Variation

Inhibition of Phagocytosis

Resistace to Oxidative Killing

Intracellular survival

Suppression of Immune Response

Fungal Immune Evasion Strategies

Antigenic variation

Alters surface proteins to escape immune recognition (e.g., Candida albicans)

Inhibition of Phagocytosis

– Capsules (e.g., Cryptococcus) and biofilms inhibit engulfment and killing.

Resistance to OXidative Killing

– Melanin and catalase enzymes neutralize reactive oxygen species.

Intracellular Survival

– Some fungi survive and replicate within macrophages (e.g., Histoplasma capsulatum).

Suppression and Immune Responses

– Fungal products can modulate cytokine production and T-cell responses

Immunosupression

Broad-spectrum Antibiotic Use

Diabetes Mellitus

Indwelling Medical Devices

Prolonged Hospitalization and ICU Stays

Environmental Exposure

Factors Predisposing to Fungal Infections

Opportunistic

Fungal Infections are often ___

Immunosupression

• HIV/AIDS (e.g., Cryptococcus, Pneumocystis).

• Organ transplant recipients (e.g., Aspergillus, Candida).

• Cancer chemotherapy and hematologic malignancies.

Broad-spectrum Antibiotic Use

Disrupts normal microbiota, allowing overgrowth of fungi like Candida

Diabetes Mellitus

Hyperglycemia impairs neutrophil function and promotes fungal growth.

Indwelling Medical Devices

Central lines, urinary catheters, prosthetics – promote biofilm-related infections

Prolonged Hospitalization and ICU Stays

Increased exposure to nosocomial fungi and invasive procedures

Environmental exposure

Construction sites (e.g., Aspergillus), bird droppings (Cryptococcus), soil (Histoplasma, Sporothrix

Proper specimen collection

Accurate fungal diagnosis begins with

refrigeration

Store at room temperature; avoid ____ of fungal samples unless otherwise specified.

selective media

Non-sterile specimens are cultured on _____ to inhibit bacterial overgrowth

Direct Microscopy

provides rapid, presumptive identification of fungal elements.

Potassium Hydroxide (KOH) Preparation

• Used to clear keratin and debris from skin, hair, or nail specimens.

• Detects hyphae, yeast cells, and spores.

• May be enhanced with DMSO (dissolves keratin faster) or Calcofluor white (fluorescent stain

Gram stain

Though primarily for bacteria, yeasts like Candida stain well and appear as Gram-positive ovoid cells, sometimes with budding or pseudohyphae.

Calcofluor White Stain

• Binds to chitin and cellulose in fungal cell walls.

• Requires fluorescence microscopy.

• Highly sensitive; used in clinical samples such as corneal scrapings or tissues

India Ink preparation

• Used to visualize capsulated yeast (Cryptococcus neoformans) in CSF.

• The capsule appears as a clear halo against a dark background

Sabouraud Dextrose Agar (SDA)

– basic medium with acidic pH to inhibit bacteria.

SDA with antibiotics (e.g., chloramphenicol, gentamicin)

suppresses bacterial contaminants.

Mycosel or Mycobiotic Agar

includes cycloheximide to inhibit saprophytic fungi.

Brain Heart Infusion (BHI) Agar

used for systemic fungi.

Cornmeal Agar

helpful in observing chlamydospore formation in Candida.

25-30C

Incubation for molds

35-37C

Incubation for yeast

2 days to 6 weeks

Incubation time ranges from ______, depending on fungal species.

Lactophenol Cotton Blue (LPCB) stain

is commonly used to visualize hyphal structures, spores, and fruiting bodies

Urease test

distinguishes Cryptococcus (urease positive) from other yeasts.

Serological and Antigen Detection Methods

These are important for rapid diagnosis, especially in systemic mycoses

Antibody detection

• Useful for histoplasmosis, coccidioidomycosis, and aspergillosis, but has limited use in immunocompromised patients.

• Methods include complement fixation, immunodiffusion, and ELISA.

Molecular techniques

provide high sensitivity and specificity, especially for fastidious or unculturable fungi.

Polymerase Chain Reaction (PCR)

• Targets ribosomal RNA genes or species-specific DNA sequences.

• Used for detection of Aspergillus, Pneumocystis jirovecii, and Histoplasma.

Real-Time PCR (qPCR)

Allows quantification of fungal DNA and can monitor fungal burden.

DNA Sequencing

ITS (Internal Transcribed Spacer) sequencing is a gold standard for fungal identification.

ITS (Internal Transcribed Spacer)

is a gold standard for fungal identification.

Next Generation Sequencing

Detects mixed fungal populations, emerging in research and epidemiology.

BSL-2 or BSL-3 Facilities

Work involving molds, especially dimorphic fungi (e.g., Histoplasma), must be conducted in