How to Detect Pneumocystis Carinii on Alveolar Exudates

Pneumocystis carinii is an opportunistic fungal pathogen, causing life-threatening pneumonia in immuno-compromised patients, particularly in AIDS patients. Infection probably occurs by inhaling the organism in aerosols. Some infections may also occur from reactivation of a latent infection. HIV-associated pneumocystis pneumonia is found in developing counties but is not major cause of death in AIDS patients as in USA, Europe, and elsewhere.

Examination of stained preparations of bronchial and alveolar washings (broncho-alveolar lavage), collected using a bronchoscope, is recommended- for the detection of Pneumocystis carinii cysts. The specimen must contain alveolar exudates if cysts are to be found. In AIDS patients, cysts can often be found also in induced sputum, i.e specimen collected after the patient has inhaled 3-5% saline mist for about 15 minutes. The hypertonic saline stimulates the coughing up of alveolar mucous materials.

            PROCESSING SPECIMEN AND CYST CONCENTRATION

  1. Add a given volume of freshly made acetyl-cysteine mucolytic reagent or pancreatin mucolytic reagent equal to twice the volume of specimen. Mix at intervals until the mucus is completely dissolved.

Caution: Specimens may contain M. tuberculosis, therefore handled the sputum with care.

 2. Centrifuge at high speed for 10 minutes to sediment the cysts. Remove and discard the supernatant fluid.

  3. Add physiological saline to the sediment, mix well, and re-centrifuge at high speed for 10 minutes. Remove and discard the supernatant fluid.

 4. Mix the sediment and make two thin preparations on slides. Allow the smears to air-dry. Fix with methanol for about 2 minutes.

Note: Because of the high prevalence of tuberculosis, prepare also a smear for Ziehl-Neelsen staining to examine for AFB.

5. Use Giemsa to stain one of the smears. Use a 1 in 25 dilution of the stain in pH 6.7 buffered water and follow the staining technique.

6. Stain the second smear with toluidine blue-0 stain

Toluidine blue stains the (cell wall) of P. carinii cysts but not the internal structures. Gieomsa stains the small internal nucleated bodies of the cysts enabling the cysts to be difference from yeast cells which may also be found in specimens.           

          An alternative staining procedure to toluidine blue-0 is the methenamine silver borate staining technique, but this is far more complex and requires a range of expensive kits. The presence of Cysts can also be demonstrated by the Papanicolaou staining procedure (mainly used in cytology laboratories).

 7. Examine the smears microscopically for an adequate length of time (cysts are often few). The cysts occur singly and in clusters.

Toluidine blue Smear: Use the 10 x and 40x objective to detect and examine the cysts (mount the smear under a cover glass using a drop of immersion oil). Look of blue staining spherical cysts about 4-7cm in diameter or cup-shaped cysts with a collapsed wall.

Giemsa stained Smear: Use the 100x objective to identify the very small purple-mauve intracystic bodies (nuclei of the trophozoities within the cyst). They can be seen in groups, up to eight in number.

The nuclei of cells of Pneumocystis carinii are best seen in thin well stained parts of the smear.