Sputum for fungi (Fungus, Yeast and Molds)

Sample

1. The sputum sample is needed to find the presence of the fungus.
2. Advise patient to get deep cough sputum.
3. The sample can be refrigerated for up to 24 hours.
4. Sputum may be stable at room temperature for 72 hours.
5. Do not freeze the sample.
6. Store it at room temperature.

Precautions

1. Sputum should be examined as fresh as possible because Histoplasma capsulatum dies rapidly at room temperature.
2. The saprophytic fungi like candida and commensal bacteria rapidly multiply if kept at room temperature and it will interfere in the separation of pathogenic fungi.
3. Try to minimize contamination with the saliva.
4. Stop aspirin, ibuprofen, naproxen, and warfarin which will thin the bronchial secretion.

Method to collect a good sample

1. Brush your teeth and rinse your mouth (don’t use antiseptic mouthwash).
2. Take a couple of long, deep breaths.
3. Breathe in deeply again and cough hard until sputum comes up.
4. Spit out the sputum into the sample cup.
5. Keep coughing up sputum until there is approximately 1 teaspoon.

Indication

1. This is done for the diagnosis of respiratory fungal infection.
2. In the case of :
   1. shortness of breath
   2. fever
   3. A  cough which is most of the time is dry.
   4. Muscle aches and pains.

Microbiology

1. Fungi are microorganisms. The majority are nonpathogenic.
2. Fungi are eukaryotic organisms and divided broadly into three main groups:
   1. Yeasts: These are single-celled. These are facultative anaerobes.
      1. Candida e.g. C.albicans
      2. Cryptococcus e.g. C. neoformans.
      3. Torulopsis.
      4. Trichosporum.
      5. Malassezia.
      6. Rhodotorula.
   2. Molds: These are filamentous fungi (hyphae). These are aerobic.
      1. These fungal hyphae form mycelium.
      2. Mycelium has two functions:
         1. The vegetative mycelium penetrates into the medium and absorbs nutrients.
         2. Aerial mycelium has reproductive structures for the spread of molds.
   3. Dimorphic fungi: This exists in both the above forms.
      1. Blastomyces dermatidis causing North American blastomycosis.
      2. Histoplasma capsulatum causing Histoplasmosis.
      3. Histoplasma duboisii causing African histoplasmosis.
      4. Sporothrix schenckii causing Sporotrichosis.
3. Some of the fungi that exist in either form are called dimorphic fungi.
4. The cell wall of the fungi is thick and composed of polysaccharides.
5. Fungi are not motile.
6. Most of these fungal diseases are opportunistic and need some predisposing factor, these are called Mycosis.
7. Predisposing factors are:
   1. Cancers.
   2. Radiation.
   3. Chemotherapy.
   4. Antibiotics.
   5. Immune deficiency syndrome.
8. The sputum fungal smear is one of the best ways to find out if your respiratory illness is caused by a fungus.
   1. A positive smear for fungus indicates a fungal infection.
9. The following types of fungal infections may be seen in the respiratory system:
   1. Aspergillosis.
   2. Blastomycosis.
   3. Coccidioidomycosis.
   4. Histoplasmosis.
   5. Cryptococcus.
   6. Pneumocystis carinii.

Normal

• A normal (negative) result means no fungus was seen in the test sample.

Abnormal Results

• When sputum is positive for fungal infection.

Procedure

1. The smear can be stained.
   1. Wet preparation:
      1. Take the skin scraping, hair, nail clipping, vaginal swab, sputum, or body fluids.
      2. Make a thin smear of sputum and mix with KOH (10%).
      3. Then smear is examined under the microscope.
   2.  Gram stain (invented by Hans Christian Gram).
      1. Make sputum smear on the slide.
      2. Fix it with heat.
      3. Stain with Gram’s stain.
      4. See under the microscope.
2. Culture on the Saubourads dextrose agar medium (SDAM).
   1. Candida albicans show raised creamy smooth colonies after 72 hours on SDAM.
   2. Other media used is nutrient agar media.
      1. Fungi grow slowly and it may take several weeks for the growth of fungi.
   3. C. neoformans form white, granular, or wrinkled first and later on moist, shiny, and mucoid colonies on SDAM.
3. Serology may be helpful in some of the fungal infections like histoplasmosis, coccidioidomycosis, and South American blastomycosis.

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