Fungal infections

Sample for fungal infections

1. The pathologist can take the sample from the patient lesion site.
2. 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. Clean the slide with 70% alcohol.
      1. Scraping should be done at the edge of the skin lesion. You can use the side of the scalpel blade or the edge of the glass 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 the outer surface.
   4. Ulcer scrapings. Avoid superficial samples, and try to get deep scraping, aspirate, or tissue biopsy are the best sample.
      1. Place the material in the saline-soaked gauze, and don’t use formaline.
   5. Pus.
   6. Cerebrospinal fluid. Collect the CSF by a spinal tap in a sterile container.
   7. Blood. Clean the site to avoid bacterial contamination. Collect the blood in special fungal media.
   8. Bone marrow.
   9. Urine. The early morning sample is the best sample, collected in a sterile container.
   10. Stool.
   11. Bronchial washings.
   12. Sputum. This sample may be collected by deep sputum, bronchoscopy, or alveolar lavage.
   13. Prostatic secretion.
   14. Tissue biopsy.
   15. Vaginal swabs are an adequate sample, and keep these in saline or transport media.
3. Blood sample for the antibody test.

Indications for fungal infections

• This procedure is done to diagnose fungal infection.

Pathology of the Fungi

Fungi are eukaryotic cells:

1.  Fungi are eukaryotic cells lacking chlorophyll, so they can not generate energy through photosynthesis.
2. The fungus nucleus contains multiple chromosomes, and the cytoplasm has mitochondria and ribosomes.
   1. Membranes surround chromosomes.
   2. It needs an aerobic environment.
   3. Most fungi are eukaryotic and divided into two main groups:
      1. Yeast.
      2. Molds.
         

         Fungus structure in detail

         

         Fungus structure

The growth pattern of fungi:

1. Many fungi grow sexually (meiosis).
2. Some 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.
5. Fungi are aerobic and can grow on simple media.

Fungi are classified into:

1. Yeast:
   1. These are round to oval unicellular fungi, which can reproduce by budding.
   2. These reproduce asexually.
   3. When budding can not separate, then it may produce pseudohyphae.
   4. Very few can form true hyphae.
   5. Yeast reproduces at a slower rate than bacteria.
   6. Examples are Candida, Cryptococcus, and Candida glabrata.

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

      Fungus filamentous shape

3. Dimorphic fungi:
   1. This exists in two from molds (mycelial form) and yeast. It depends upon environmental conditions and temperature.
   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.
      

      Fungus dimorphic

4. Spores: It is reproducing bodies of molds.
   1. Spores are rarely seen in skin scrapings.
5. Molds: These are also called mycelia.
   1. There are multicellular colonies composed of clumps of intertwined branching hyphae.
   2. Molds grow by longitudinal extension and produce spores.
6. Saprophytes: These fungi utilize organic matter like rotten soil vegetation as an energy source.

Fungal infections are classified into:

1. Superficial infection:
   1. The mucosa leads to Thrush in the mouth by yeast.
   2. Filamentous fungi, e.g., ringworm, cause nails, skin, and hair infection.
2. Deep or systemic fungi:
   1. Deep or systemic fungal infections are characterized by the involvement of visceral organs or penetrating type of infection.
   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 due to their toxic metabolites, e.g., aflatoxin.
3. 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) sees fungus directly 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 underneath 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, you can see fungal elements like spores or mycelia.
4. Stained smears:
   1. The clinical sample is stained with gram stain or Papanicolaou stain.
   2. Wright or Giemsa stain is used for Histoplasmosis.
   3. The advantage is that you can get results immediately.
5. 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.
6. 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 antifungals will prevent 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.
7. 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 infections.
   2. With a titer greater than 1: 32 indicates 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.
8. 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 is 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 is where there is the involvement of deeper tissues and organs.
   1. Examples are:
      1. Blastomycosis (Blastomyces dermatitidis).
      2. Histoplasmosis (Histoplasma capsulatum).
      3. Coccidioidomycosis (Coccidioides immitis).
      4. Cryptococcosis (Cryptococcus neoformans).
      5. Paracoccidioidomycosis.
         

         Skin fungal diseases

         

         Fungus shapes

         

         Fungus septate and non-septate hyphae

Treatment of Fungal infections

1. Clotrimazole or Miconazole can treat superficial infections.
2. A local application like Benzoic acid was also used.
3. For severe infection, oral Griseofulvin was 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.
5. For systemic fungal infection, drugs used are:
   1. Amphotericin B.
   2. Itraconazole.
   3. Ketoconazole.
6. For cutaneous fungal infections, drugs used are:
   1. Griseofulvin.
   2. Locally used are nystatin, clotrimazole, and miconazole.

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