Fungus, Fungal infections, Diagnosis of Fungal infection

Sample

• The sample can be taken by the pathologist from the patient lesion site.
• The sample can be taken from the following sites.
  1. SKIN: Scrape the area with a glass slide or knife and get an adequate amount of scrapings on the slide.
  2. HAIRs: Pluck the hairs with tweezer with roots because the fungus is deep-seated and is near the roots of hairs.
  3. NAILS: Get from the nail undersurface areas and not from the outer surface.
  4. Ulcer scrapings.
  5. Pus.
  6. Cerebrospinal fluid.
  7. Blood.
  8. Bone marrow.
  9. Urine.
  10. Stool.
  11. Bronchial washings.
  12. Sputum.
  13. Prostatic secretion.
  14. Tissue biopsy.
• Blood sample for the antibody test.

Indication

• This procedure is done to diagnose fungal infection.

Pathology

1. Fungi are eukaryotic. The fungus nucleus contains multiple chromosomes and the cytoplasm has the mitochondria and ribosomes.
   1. Chromosomes are surrounded by membranes.

2. Growth pattern:
   1. Many fungi grow sexually (meiosis).
   2. Some of the fungi grow in filaments (hyphae) and form a mesh called Mycelium.
   3. Some of the fungi produce buds like yeast is an exception.
   4. Few of these are pathogenic.
3. Fungi are aerobic and can grow on simple media.
4. Fungi are classified into:
   1. Yeast
      1. These are round to oval unicellular fungi and these can reproduce by budding.
      2. These reproduce asexually.
      3. Few may produce pseudohyphae.
      4. Very few can form true hyphae.
      5. Examples are Candida, Cryptococcus, and Candida glabrata.

1. 2. Filamentous fungi.
      1. These are a group of fungi that cause the infection of skin, nails, and hairs.
      2. These fungi do not involve living tissue.
      3. These are Trichophyton, Microsporum, and Epidermphyton.

1. 3. Dimorphic fungi.
      1. This exists in two from as molds (mycelial form) and yeast.
      2. The yeast form is found in the infected tissue and on artificial media at 37 °C.
      3. Filamentous fungi are present in the soil and grow on artificial media at 22 to 25 °C.
      4. These are Blastomycosis, Coccidodioides, Histoplasmosis, and Sporothrix.

5. Fungal infections are classified into:
   1. Superficial infection:
      1. The mucosa leading to Thrush in the mouth by yeast.
      2. The infection of the nails, skin, and hairs is caused by the filamentous fungi e.g ringworm.
   2. Subcutaneous infection is caused by the trauma and gives rise to the sinus.
   3. Systemic infections (mycosis) are serious and fatal for the patient.
   4. Mycotoxicosis is due to eating the infected food by these fungi and is due to their toxic metabolites e.g. is due to aflatoxin.
   5. An allergic reaction like type I and type III is mostly due to fungal spores.

Methods to identify the fungus

1. Wood’s light (Ultraviolet rays) to see directly fungus over the hairs.
   1. Then it is examined in the darkroom where infected hair gives fluorescence of bright yellow-green light.
2. Phase-contrast microscopy:
   1. This is quick and no need for any special stains.
   2. There is a very good identification of fungi.
3. Direct microscopic examination:
   1. Clean the skin with 70% ethyl alcohol.
   2. The different sites are:
      1. Make scrapings from the edge of the lesion.
      2. Pluck the hairs.
      3. Take a sample from the underneath of the nails, not the outer surface.
   3. Procedure :
      1. Add  Potassium hydroxide (KOH) 10 to 20% solution.
      2. Leave this scraping for 10 to 20 minutes for the keratin to be digested.
      3. Cover with coverglass and can press it to make a thin smear.
      4. Then under the microscope can see fungal elements like spores or mycelia.
4. The culture of various fluids:
   1. Blood culture: Blood is collected in the special fungal culture media.
   2. Urine culture: Collect the morning sample which has a good yield.
   3. Cerebrospinal fluid can be collected for the culture.
   4. Sputum culture: The sample can be obtained by bronchoscopy or alveolar lavage.
5. Culture can be done to identify the fungus on Sabouraud’s media. This is good for the cutaneous or vaginal sample.
   1. Add antibiotics like chloramphenicol to suppress the growth of bacteria (selective media).
   2.  Brain-heart infusion media with antibiotics and antifungal will prevents bacterial growth and the rapidly growing fungi.
      1. The fungi are a slow-growing organism.
      2. Examine daily up to 21 days and daily check the growth.
      3. Identify the colony’s appearance and pigmentation.
   3. Transfer the colony to slide and stain with lactophenol cotton blue and cover with the cover glass.
      1. Examine under the microscope.
6. Serological test:
   1. These are not highly reliable.
      1. Antibodies are present in 70 to 80% of the patients.
      2. There may be cross-reaction of the antibodies.
      3. These serological tests cannot tell recent or old infection.
   2. with a titer greater than 1: 32 indicate a fungal infection.
   3. A complement test can be done for the diagnosis of Histoplasmosis and coccidioidomycosis.
   4. The immunodiffusion test is helpful for the diagnosis of Blastomycosis.
7. Biopsy of the tissue can be stained with:
   1. Periodic acid-Schiff (PAS) reaction.
   2. Silver stain (Gamori’s methenamine silver).

Types of Fungal diseases

1. Dermatophytosis where there is the involvement of superficial skin e.g. Athlete’s foot, and Ringworm.
   1. Other examples are:
      1. Tinea versicolor.
      2. Tinea nigra.
      3. Piedra (white piedra).
      4. Black Piedra.
2. Subcutaneous mycosis where there is the involvement of deeper tissue and muscles.
   1. These will give rise to chronic granulomatous inflammation.
   2. Sporotrichosis.
   3. Chromomycosis.
   4. Mycetoma.
3. Systemic mycosis where there is the involvement of deeper tissues and organs.
   1. Examples are:
      1. Blastomycosis.
      2. Histoplasmosis.
      3. Coccidioidomycosis.
      4. Paracoccidioidomycosis.

Treatment

1. The superficial infection can be treated by the Clotrimazole or Miconazole.
2. A local application like Benzoic acid was also used.
3. For severe infection, oral Griseofulvin used for 4 to 6 weeks.
   1. In the case of hairs used for 3 to 6 months.
   2. In the case of nails for one year.
4. Oral Terbinafine and Itraconazole are also effective.

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