Introduction to Mycology

Basic Mycology Overview

• Mycology is the study of fungi, which are ubiquitous in nature
• More than 200,000 species of fungi have been identified
• Approximately 400 fungi species are considered medically important
• Fungi are facultative parasites, meaning they can be part of normal flora
• Most fungi are obligate aerobes, while some are aerotolerant
• All fungi are chemotropic and cannot perform photosynthesis
• Fungi secrete substances (enzymes and toxins) to degrade organic matter, contributing to their saprophytic nature

Terminologies

• Key terms to understand include yeast, mold, spore/condidia, dematiaceous, dimorphic fungi, pseudohyphae, hyphae/mycelia, and germ tube

Yeast vs. Mold Characteristics

• Yeast are unicellular; molds are multicellular
• Yeast and mold both have cell walls made of chitin, glucan, and mannan
• Some molds have melanin in their cell walls, giving them a dematiaceous appearance
• Both yeast and mold have cell membranes containing ergosterol
• Some, but not all, yeasts have capsules, while molds do not
• Yeast form pseudohyphae in survival mode
• Some yeast can convert into mold
• Molds have rodlets, giving it a stable and sturdy protein structure outside of the cell wall
• Molds have true hyphae

Yeast Cell Wall

• When without a capsule
• Consisting of Mannoproteins, B-1,3-glucan, B-1,6-glucan and Chitin
• Influences virulence and resistance

Yeast Cell Wall

• When with a capsule
• The capsule is located outside of the cell wall
• Hides fungi from immune response

Mold Cell Wall

• Molds are generally more invasive than yeast
• Galactosaminogalactan, B-1,3-glucan, B-1,3-1,4-glucan, B-1,6-glucan ,α-1,3-glucan, Galactomannan, Melanin, Rodlet, and Chitin

Yeast Cultures

• Grown at 37 degrees Celsius
• Resembles bacterial cell macroscopically but with distinctive odor
• The shape is defined as round or oval microscopically
• Reproduce mostly by budding cell (blastoconidia)
• The species Candida albicans produces pseudohyphae

Mold Cultures

• Grow better at room temperature
• all have hyphae, which can be septate or aseptate
• Produce mycelia on culture, differing macroscopically between species
• Reproduce mostly by condidia (or spore)

Dimorphic Fungi and Germ Tubes

• Dimorphic fungi can convert between yeast and mold forms depending on environmental conditions like temperature and nutrition
• Germ tube is a specific structure facilitating this conversion

Pseudohyphae vs. Germ Tube

• Pseudohyphae are elongated budding cells failing to detach from their mother cell
• Germ tubes appear after incubation in mammal serum at 37°C

Basics of Fungal Identification

• Fungal identification includes Staining (KOH, LPCB), Culture on Sabouroud Dextrose Agar, Serology (Antigen/Ab testing), and NA based (PCR, sequencing)
• Staining is easy and fast with lowest price

KOH vs LPCB Staining

• KOH (Kalium hydroxide, 10%) has protease activity to destroy tissue, while LPCB (Lactophenol cotton blue) contains phenol to inactivate fungi
• KOH is recommended for yeast or direct clinical specimens, whereas LPCB is recommended for molds but not direct clinical specimens
• KOH gives a transparent result (with a slight green hue), and LPCB gives a blue-colored structure (except for dematiaceous fungi) under the microscope KOH
• 10% KOH is used to prepare the specimen
• Add one drop of KOH 10% onto object glass
• Take the specimen using sterilized loop
• Make a suspension with KOH 10%
• Cover the suspension with coverglass
• Gently heat the specimen
• The the specimen should be identified within 10 minutes.
• Clinical specimen probably needs longer KOH exposure, depending on the thickness.
• Obeserve presence of budding cell, shape of budding cell and the presense of pseudohyphae (usually only in clinical specimen) LPCB
• Add one drop of LPCB onto object glass
• Take the specimen using sterilized loop
• Make a suspension with LPCB
• Cover the suspension with coverglass
• LPCB does not have protease activity, thus cannot be used for examining fungal presence from clinical specimen.
• Note that LPCB does not need any heating.
• The species should exhibit presence of hyphae, determine whether the hyphae is septate hyphae or aseptate hyphae, presense and shape of conidia and location of conidia (free, inside macroconidia, etc)

Fungal Culture Methods

• Involve use of Sabouroud Dextrose Agar (SDA).
• Potato Dextrose Agar, cornmeal agar, and cooked rice media may also be used
• Fungal cultures must be incubated for 2-6 weeks in both temp (37oC and RT) before negative conclusion, especially when mold infection is suspected
• Fungal culture identification includes upper, reverse observation, and microscopic identification
• Upper and reverse observation include recording the surface color and type of colony

Upper Side Observations in Fungal cultures

• Glabrous colonies are smooth
• Velvety colonies look like carpet with short aerial hyphae
• Cottony colonies look like cotton, aerial hyphae is easily seen; and Fluffy colonies look like fluff with voluminous aerial hyphae
• Presence of groove is recorded
• If there is a presence of diffusible pigment that is recorded

Rationale for Fungal Identification

• There could be varied microscopic and macroscopic appearance between several fungi
• Information about body site and patient history should be included during identification
• Remember that fungi are mostly facultative parasites.

Serology

• Most kits detect the presence of specific antigens (e.g., beta-d-glucan in Candida albicans, galactomannan in Aspergillus spp.)
• In immunocompetent patients, serological results have higher value than in immunocompromised patients
• Since fungi have diverse characteristics (niches, geographical influence), serologic tests need evaluation of sensitivity and specificity for each population

Nucleic Acid-Based Tests

• Conventional and real-time PCR identify the presence of a conserved part inside a nucleic acid strand
• Whole genome sequencing records the nucleic acid sequence to detect mutations or genomic evolution

Mycosis

• Mycosis is any infection caused by fungi
• It is highly associated with hygiene, personal habits, and immune system strength
• In immunocompetent patients, mycosis generally resolves itself
• However, in immunocompromised patient, myosis can lead to systemic infections

Skin Structure

• Superficial Mycosis effects the Stratum corneum, the outermost layer of skin
• Cutaneous mycosis effects skin's epidermis,
• Subcutaneous mycosis effects the dermis

Types of Mycosis

• Superficial: limited to keratin layer of stratum corneum.
• Does not progress to systemic and related with hygiene and personal habit 1)
• Cutaneous: Limited to epidermis. 1)and/or presence of risk factor
• No Patchy, itchy lesion
• Subcutaneous: Limited to dermis, may progress to systemic. Usually caused by direct inoculation due to trauma
• Forming mass consisting of fungal component inside dermis. May enter vascular / lymphatic vessel
• Systemic: Involving many sites within the body. Always associated with poor immune system

Summary

• Fungi is mostly part of normal flora
• Mycosis always has underlying factor
• The types of fungi are yeast and mold
• Yeast - budding cell and pseudohyphae
• Mold - (true) hyphae and conidia
• Identification - staining (KOH, LPCB), culture (ie Sabouroud Dextrose Agar), serology, and nucleic acid-based test.
• Fungal identification must consist of microscopic and macroscopic observation
• The types of mycosis is superficial, cutaneous, subcutaneous, and systemic