Fungal Specimen Collection and Identification Techniques
Fungal Specimen Collection and Identification
Hairs: From scaly areas of alopecia, hairs should be pulled out with the root using sterile forceps and suspended in a sterile petri dish or tube.
Nails: Nail-infected samples are taken from under the nail to obtain subungual soft material. Fine nail fragments can be collected using a scalpel or scissors. Nail clippings can be isolated for dermatophyte or Candida identification.
Exudates: Disinfect the skin overlying the lesion. Pustular exudates should be aspirated with a sterile needle and syringe and transported in the same syringe.
Sputum: The first sample should be collected in the morning before breakfast. Rinse the mouth with water before coughing. Place the sample in a sterile petri dish.
Feces should be deposited directly into a sterile container for stool samples, or a rectal swab can be used.
Liquid CSF: For fungal cultures, a sufficient amount of CSF is required. Store at room temperature in a sterile tube.
Blood: Directly inoculate 10 to 20 ml of citrated or heparinized blood into broth or onto Sabouraud glucose agar with 1% chloramphenicol.
Tissue Biopsy: Biopsies of tissue from the site of infection should be transported in sterile gauze moistened with sterile saline in sterile packaging. Take care to avoid dehydration and never freeze the sample.
Identification of Dermatophytes
Macro and microscopic characteristics in culture isolation need to be studied.
Cryptococcus
There are 16 species, but the only pathogenic species is C. neoformans.
Direct Examination:
For direct examination, take exudate or sputum. In the case of CSF or urine, the sample should be centrifuged. Perform India ink staining diluted in water (1:5). Cryptococcus yeast is easily demonstrated as 4 to 8 μm in diameter, surrounded by a mucoid capsule 1 to 10 μm thick that is not stained with ink, resembling a clear space. Occasional pseudohyphae may be present.
In humans, ringworm and candidiasis are frequently encountered diseases, though underreporting of cases is common.
The main feature of fungi is the absence of chlorophyll, so they cannot perform photosynthesis, and their food source is organic matter. Other common features are that they are heterotrophic eukaryotes, possess a chitin wall, absorb nutrients, and present a thallus or mycelium. They reproduce by spores that germinate into long filaments called hyphae. The thallus or mycelium that penetrates inside the substrate is called vegetative mycelium, which ensures development, nutrition, fixation, and building of the reproductive part. The thallus forms the reproductive organs and can be represented by filamentous fungi, yeasts, or pseudohyphae.
If the thallus is dissociated, yeast colonies produce rapid growth, which is creamy and can be replated like bacteria. If the thallus is filamentous, colonies result in mold growth with centrifugal air intermingled with relatively long filaments or grouped in a compact manner. Growth is slower than that of opportunistic fungi.
Fungi Producers of Superficial Mycoses
These fungi belong to the dermatophytes, Pityrosporum genus, and Candida species, causing skin conditions. They are characterized by invading the dead keratin of skin, hair, nails, fur, feathers, hooves, and horns.
Their identification is based on observation of the fungus in the lesions and the macroscopic and microscopic morphological characteristics of cultures.
Dermatophytes
There are three kinds of fungi:
| Genus | Skin | Hair | Nails |
|---|---|---|---|
| Epidermophyton | + | – | + |
| Trichophyton | + | + | + |
| Microsporum | + | + | – |
Mycological Techniques
A. Sampling
Clean the area of the lesion with a soft sponge soaked in 70% ethanol to remove surface contaminants.
In order to request a review, indicate the patient’s personal history, medical history, and geography. Obtain a sufficient sample in sterile plates without antifungal therapy, or after a minimum suspension of 3 days.
General Considerations by Type of Sample
Skin: For dry skin mycoses, scrape the periphery of the lesion with a sterile lancet to collect flakes in a sterile petri dish.
If the lesion is wetter, obtain a sample with a sterile swab soaked in sterile saline, scraping the area and referring it immediately to the laboratory. If you suspect Pityriasis versicolor, sample with transparent tape over the injury and affix it onto a glass slide for direct microscopic examination.