A Guide to the Study of Basic Medical Mycology
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A Guide to the Study of Basic Medical Mycology
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| ISBN-13: | 9781482824124 |
|---|---|
| Publisher: | Partridge Singapore |
| Publication date: | 09/11/2014 |
| Pages: | 92 |
| Sales rank: | 880,434 |
| Product dimensions: | 8.50(w) x 11.00(h) x 0.24(d) |
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A Guide to the Study of Basic Medical Mycology
By Kee Peng Ng, Tuck Soon Soo-Hoo, Shiang Ling Na
PartridgeSG
Copyright © 2014 Kee Peng Ng, Tuck Soon Soo-Hoo, Shiang Ling NaAll rights reserved.
ISBN: 978-1-4828-2412-4
CHAPTER 1
Section 1. Introduction to Basic Mycology
Fungi include two diverse forms: moulds and yeasts.
A yeast colony is usually single, round, raised, or convex. The colony may be white, red, or black in colour (Fig. 1A). The vegetative structure of yeast is single, unicellular cells, 4-8 µm in diameter with buddings (Fig. 1B). The yeasts may produce pseudohyphae or true hyphae (Table 1) and reproduce by budding.
A mould colony is fuzzy or cottony in appearance (Fig. 2A). A single growing vegetative structure of mould is known as a hypha or hyphae (more than one hypha). The hyphae can be nonseptated (Fig. 2B) or septated and branched (Fig. 2C). The mycelium growing on the surface of the agar medium is called aerial mycelium, and those growing down into the agar are called vegetative mycelium. A mould colony is therefore made up of aerial and vegetative mycelia, which are referred to as thallus (thalli).
Most of the fungi isolated in the clinical laboratory produce only asexual spores. Identification of moulds is made based on the characteristics of the spores and their method of reproduction.
A number of moulds reproduce as yeast-like cells on enriched media when incubated at 37°C or in tissue. The fungi are in mould form on SDA (Sabouraud Dextrose Agar) when incubated at a lower temperature, e.g. 30°C. This phenomenon of changing vegetative structures is called dimorphism. As temperature is a critical factor in the formation of dimorphic characteristic, the phenomenon is also called thermal dimorphism.
Classification of fungi
There are four recognized divisions in the kingdom of fungi: Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota. Members of Chytridiomycota are plant pathogens.
The anamorphic fungi or fungi imperfecti (Deuteromycota) refer to groups of fungi not included in these divisions. These fungi only reproduce asexually without a sexual reproductive stage (telemorph). Some of the fungi in the groups that have telemorphs are placed in the Ascomycota or Basidiomycota.
The anamorphic fungi are artificially classified according to the vegetative form of growth and the characteristic production of asexual spores. These fungi are classified into Blastomycetes, Hyphomycetes, and Coelomycetes (Table 2).
The Hyphomycetes produce no fruiting body, and the hyphae are septated. The arrangement and size of conidia depend on the species. Coelomycetes have septate hyphae, and the conidia are produced in fruiting bodies called pycnidia or acervuli. Blastomycetes are yeast-like fungi.
Most of the medically important fungi are within the Blastomycetes, Hyphomycetes and Zygomycetes. The key features listed in Table 3 can be used as a simplified guide to identify the major groups of fungi frequently isolated in a routine clinical mycology laboratory.
Characteristics of fungi
Yeasts
The colonies are single, round, and moist-to-waxy. The cells are predominantly unicellular; they may be spherical, elliptical, or cylindrical. They may be variable in size, ranging from 4-8 µm.
On Cornmeal-Tween 80 agar, many types of yeasts produce pseudohyphae, but a number of yeast-like fungi produce true hyphae.
On a gram stained smear, the yeast cells may be spherical or cylindrical with a predominance of budding cells (Fig. 3A, B). The new cell is abstracted from the mother cell and then enlarged to form a matured cell.
Moulds
The colonies are made up of hyphae and grow by elongation at the tips or by lateral branching, forming a tangled mass of hyphae called mycelium (Fig. 4A, B). The hyphae may become specialized cells that produce conidia called conidiogenous cells.
The conidiophores are morphologically distinct from the vegetative hyphae.
In some fungi, e.g. Acremonium, the conidiophores and conidiogenous cells are actually one and the same structures.
In Aspergillus or Penicillium, conidiogenous cells and conidiophores are two distinctly different structures.
Describing the colonial morphology
All medically important fungi grow on Sabouraud Dextrose Agar (SDA). The description of the morphological characteristics of fungi is normally based on the cultural appearance on SDA incubated at 30ºC.
1. Rate of growth: a rapid grower produces characteristic morphology on SDA within five days. A moderate grower produces characteristic morphology between five to ten days, while a slow-growing fungus may take two to three weeks to develop a characteristic morphology.
2. Texture: describing the height of the aerial mycelium (Fig. 5).
• Yeast-like: colony solid, no production of aerial mycelium (Fig. 5A).
• Cottony or woolly: fungus produces highly dense aerial mycelium (Fig. 5B).
• Velvety: colonies produce a low, compact aerial mycelium (Fig. 5C).
• Granular or powdery: colonies are flat and crumbly with dense conidia production. The granular texture is rough, like granulated sugar; the powdery texture is like flour (Fig. 5D).
• Labrous or waxy: colonies are smooth, produce no aerial mycelium.
3. Topography: describes the physical appearance of fungal cultures (Fig. 6).
• Flat with radial folds (Fig. 6A).
• Rugose: colonies have deep furrows, irregularly radiating from the centre with entire edge (Fig. 6B).
• Umbonate: colonies possess a button-like central elevation. They may be accompanied by rugose furrows around the button. The edge may be entire or scalloped (Fig. 6C).
• Verrucose: colonies exhibit a wrinkled, convoluted surface (Fig. 6D).
• Cerebriform: colonies exhibit many brain-like folds (Fig. 6E).
It is important to describe the topographic characteristic of the reverse side of the culture plate like the colour and the presence of radial folds or ridges (Fig. 6F).
4. Colour: the pigmentation of the colony is variable depending on cultural conditions. The pigmentation of the culture is related to the pigmentation of the spores or vegetative hyphae (Fig. 7).
• The colour on the reverse colony is the soluble pigments produced by the fungus diffused into the medium and the original pigmentation of the hyphae.
The type of spores
Most clinically important fungi belong to fungi imperfecti and produce only asexual spores.
The spores (also called conidia) are produced by segmentation or budding of the tips of the hyphae or from the walls of hyphae. The conidiogenous cell is the hyphal structure that produces conidia.
a. Arthroconidia
• The fragmentation of hypha forms a chain of uniformly sized asexual spores (Fig. 8A).
• These conidia may be randomly arranged (Fig. 8B) in the hyphae or separated by a sterile cell called a disjunctor, e.g. Geotrichum, Trichosporon, Hendersonula toruloidea, Malbranchea.
b. Chlamydospores
• They are produced by the cells of pseudohyphae or true hyphae of many filamentous fungi or Candida species.
• They are resting spores, thick-walled and heat resistant, and can be terminal (form at the end of hyphae, Ch) (Fig. 9A) or intercalary (form in the hyphae, In) (Fig. 9B).
c. Blastospores (Bl)
• These are formed by budding. The young daughter cell is abstracted from the parent cell and then enlarges itself to form a new mature cell (Fig. 10).
d. Aleuriospores
• These can be sessile or lateral aleuriospores that develop directly on the vegetative mycelium called conidiophore. The phialides produce aleuriospores which can be single-celled (microaleuriospore) or multi-celled (macroaleuriospore).
The conidiophore of Aspergillus arises from a basal cell on the hypha and enlarges at the end to form a vesicle. Flask-shaped phialides develop over the surface of the vesicle, and conidia are produced successively from the tip of the phialides (Fig. 11). The spores are usually formed in chains. The phialides may be borne directly on the vesicle (uniseriate) (Fig. 12A) or may be borne on prophialides that arise from the vesicle (biseriate) (Fig. 12B).
The terminal portion of conidiophore of Penicillium branches into finger-like projections called metulae from which flask-shaped phialides are formed. Conidia (aleuriospores) are abstracted from the tip of the phialides in chains. The conidiophore can be monoverticillate (unbranched), biverticillate, or terverticillate (Fig. 13).
The spores of dematiaceous fungi are formed at the enlarged tip of a stalk-like conidiophore or phialides. These spores bud to form spores at their distal end. The process continues until branching chains of conidia are formed with the youngest spores at the end.
The types of sporulations in dematiaceos fungi:
1. Phialophora-type sporulation: a flask-shaped phialide with the terminal end having a flared lip around the opening from which conidia are extruded (Fig. 14A).
2. Rhinocladiella-type sporulation: a conidiophore that develops terminally or along mycelium, the conidiophore develops into a swollen roughened appearance from which conidia develop along the length and terminus of the conidiophore (Fig. 14B).
3. Cladosporium-type sporulation: conidiophore with terminal or lateral ramifications producing branched conidial chains (Fig. 14C).
In Zygomycetes: sporangiospores (endospores) are developed inside a swollen, cellophanelike structure called a sporangium that is developed on a hypha, or a branch called a sporangiophore (Fig. 15).
CHAPTER 2Section 2. Laboratory Diagnosis of Fungal Infections
Sample collection
• All samples for mycological investigation must be collected in a sterile container.
• The type of specimen depends on clinical presentation. It can be skin scraping, nail clipping, hair, sputum, tissue or caseous material, body fluids, or urine.
• For fungal blood culture, the blood is collected in commercially prepared blood culture bottles.
Direct microscopic examination
• Clinical samples are treated with 40% KOH that causes keratin cells to swell and reveal the fungal elements in the skin or nails (Fig. 16).
Stains commonly used in the Mycology laboratory
• Lactophenol Cotton Blue stain: Cotton Blue is an acid dye that stains the chitin in fungal cell walls.
• India ink: India ink contains carbon black particles that block out all the light except for the polysaccharide coating produced by fungi or bacteria. This is used in the mycology laboratory principally for identifying the capsule of Cryptococcus neoformans (Fig. 17A).
• Gomori's methenamine silver nitrate stain (GMS): This causes the cell wall to appear dark due to the deposit of silver stain (Fig. 17B).
Cultural procedures
Morphology is primarily used to establish the genus of the fungus; some fungal isolates can be identified to the species level.
Sabouraud Dextrose Agar (SDA) is the culture medium commonly used in the laboratory. The plate may have to be covered with olive oil if Malassezia furfur is suspected.
The wet-mount or tease-mount technique is widely used in the mycology laboratory for preparing fungal (mould) colonies for microscopic examination.
The slide-culture technique (Fig. 18) permits the microscopic observation of the undisturbed arrangement of spores and hyphae. This technique is important for the identification of filamentous fungi but not yeast-like fungi.
A. Laboratory procedures for the identification of yeast-like fungi
The laboratory algorithm for identification of yeast-like fungi is different from that for moulds. For identification of yeast-like fungi, potato dextrose agar or Cornmeal-Tween 80 agar may be needed to stimulate the production of chlamydospores or blastospores.
• Describe the characteristics (colour, texture, and topography) of the colony (Fig. 19).
• Confirm the yeast-like cells by performing a gram stain (Fig. 3).
• Differentiate Candida species and Cryptococcus species by urease test.
• A urease test negative is most likely Candida species; further identification of the Candida species is described in the respective section.
• A urease test positive could be Cryptococcus or Trichosporon species. Refer to the respective sections for further identification.
B. Laboratory procedures for the identification of moulds
Both a wet mount and slide-culture are required for the microscopic examination of a fungal culture.
• Describe the cultural characteristics (colour, texture, and topography) of the colony (Fig. 20).
• Prepare a Lactophenol Cotton Blue wet mount to study the microscopic morphologies of the moulds.
• Prepare a slide-culture for better demonstration of the microscopic features (Fig. 21).
Biochemical tests
Biochemical tests are used to differentiate the yeast-like fungi, especially the Candida and Cryptococcus species.
The carbohydrate assimilation test is based on the capability of the yeasts to assimilate the different types of carbon sources.
The carbohydrate assimilation test can be prepared in the laboratory using carbon-free base agar and filter paper discs impregnated with various carbohydrates (Table 4). The organism is cultured on the carbon free base agar with carbohydrate discs and incubated at 30°C for 24-48 hours (Fig. 22).
The carbohydrate assimilation test can be performed using the commercial API 20 C AUX system. The system consists of 20 cupules containing dehydrated substrates, which enable the performance of 19 assimilation tests. The cupules are inoculated with a semi-solid minimal medium, and the yeasts will only grow in cupules if they are capable of utilizing each substrate as the sole carbon source.
The reactions are read by comparing them to the controls, and identification is obtained by referring to the analytical profile index or using identification software.
Molecular identification of fungus
The molecular identification approach, particularly Polymerase Chain Reaction (PCR), is now widely used in the laboratory as an additional method to complement phenotypic or morphological approaches in accurate fungal species identification. The International Sub-commission on Fungal Barcoding has proposed the ribosomal Internal Transcribed Spacer (ITS) region as the prime fungal molecular marker for species identification. Many other markers have been designed and extensively used for specific purposes such as: subunit of RNA Polymerase genes RPB1 & RPB2, D1/D2 large subunit rDNA gene, and β-tubulin.
The workflows of ITS1-5.8s and RNA-ITS2 ribosomal RNA regions in fungal identification include harvesting the fungal culture on a medium, DNA extraction, PCR amplification, gel electrophoresis of amplified DNA (Fig. 23) followed by subsequent amplicon purification. Purified amplicons are subsequently prepped for cycle sequencing where sequencing primers, deoxynucleosidetriphosphates (dNTPs), and DNA polymerase are linearly amplified and subjected to dye terminator sequencing (Fig. 24).
Raw data from the process of dye terminator sequencing in the form of chromatograms are subjected to quality assessment and trimming to eliminate sequencing errors using suitable cutoff values. Cleaned amplicons from corresponding primer pairs are then assembled and subsequently subjected to phylogenetic analysis. This analysis involves data mining of suitable comparative species from publicly available database such as the NCBI to obtain a list of possible IDs based on the similarity of the unknown isolate to the reference sequences on the NCBI database. Data-mined datasets should be aligned using multiple sequence alignment tools such as Clustal W, Clustal X, or MUSCLE.
Phylogenetic tree (Fig. 25) construction using the appropriate substitution models and matrices is conducted using the finalized alignments to obtain the taxonomic position of the species.
Histopathology examinations
Histopathology is an important diagnostic tool in mycology. It is cheap and provides a rapid presumptive identification of the infecting fungus. Some fungi cannot be cultivated in the laboratory, e.g. Rhinosporidium seeberi and Pneumocystis jirovecii. The diagnosis of the infections is derived from the histopatholgical examination of clinical specimens.
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Table of Contents
Contents
Section 1. Introduction to Basic Mycology, 1,Section 2. Laboratory Diagnosis of Fungal Infections, 14,
Section 3. Yeasts and Yeast-like Fungi, 26,
Section 4. Hyaline Hyphomycetes, 2,
Section 5. Dermatophytes, 59,
Section 6. Zygomycetes, 63,
Section 7. Dimorphic Fungi, 67,
Section 8. Dematiaceous Hyphomycetes, 70,
Section 9. Non-cultivable Fungus, 83,