Pneumocystis jirovecii in Giemsa Stain and Its Details
Pneumocystis jirovecii in Giemsa Stain
A cyst of Pneumocystis jirovecii in Giemsa stain showing internal nuclei up to 8 in number as shown above image. Giemsa stain comes under a type of Romanowsky stain. The name of this stain has come from the surname of a German chemist Gustav Giemsa, who created a dye solution. It was initially designed for the detection of malarial parasites in blood smears, but it is also used in histology for routine examination of blood smears. This technique uses for the demonstration of other than malarial parasites, microorganisms like Helicobacter pylori,
Chlamydia trachomatis, Borrelia species, Histoplasma capsulatum, Pneumocystis jiroveci, Penicillium marneffei and occasionally bacterial capsules and parasites like Toxoplasma gondii, Leishmania donovani , Giardia lamblia, etc. It is also applied to differentiate nuclear and cytoplasmic morphology of the various blood cells like RBCs, WBCs, and platelets.
In cytogenetics, it stains the chromosomes and identifies chromosomal aberrations.
a) Preparation of Giemsa stain
- Giemsa stock powder : 1 gm
- Glycerin : 54 ml
- Methanol : 84 ml
Giemsa powder is mixed in 54 ml of glycerin and pre-heated up to 60°C.
Then add methanol, shake the mixture and allow to stand for 7 days.
Filter before use.
b) Buffer solution (stock)
- Potassium dihydrogen phosphate: 2.72 gm
- Distilled water: 100 ml
- Sodium hydroxide: 0.8 gm
- Distilled water: 100ml
Dissolve both powders in distilled water.
50 ml of potassium dihydrogen phosphate is mixed with 23.6 ml of sodium hydroxide.
The pH of the solution is adjusted to 6.8.
Working Giemsa Stain Solutions
- Giemsa stock: 10 ml
- Working buffer: 90 ml
Should be prepared fresh then use.
Buffer solution
- Stock buffer: 20 ml
- Distilled water: 480 ml
Principle of Giemsa stain
Giemsa contains Methylene blue(AzureII)/Eosin. Methylene blue on oxidation produces colored compounds termed ‘Azure’ that have the ability to combine with Eosin. Methylene blue azure is blue-violet and stains acidic cell components while eosin is red and stains basic cell components.
Procedure of Giemsa stain
For bone marrow imprints and smears
Smears are fixed in methanol for 30 minutes.
Smears are stained in working Giemsa solution for 20 minutes
Wash under running tap water for 5 minutes.
Air dry smears, clear in xylene and mount in D.P.X.
For Paraffin Section
De-paraffinize and hydrated sections to tap water.
Flood slide with Giemsa stain for 15-20 minutes.
Wash in tap water.
Differentiate 0.2% acetic acid 1 dip.
Wash in running tap water.
Dehydrate, clear and mount with D.P.X
Result interpretation of Giemsa stain
Nuclei: Blue
Cytoplasm: Pink
H. pylori and L.D bodies: Blue
Mast cell: Magenta pink
Tissue elements: Shades of blue to pink
Collagen, Muscle, and Bone: Pale pink
Erythrocytes: Salmon pink
Malaria parasite: Malaria parasites have a red or pink nucleus and blue cytoplasm
Borrelia spirochetes: Mauve-purple
Chlamydia trachomatis inclusion bodies: Blue-mauve to dark purple depending on the stage of development
Introduction of Pneumocystis pneumonia (PCP)
Pneumocystis pneumonia is a serious fungal infection which the causative agent Pneumocystis jirovecii. PCP spreads from person to person through the air. Some healthy adults can carry the Pneumocystis fungus in their lungs without having symptoms, and it can spread to other people, including those with weakened immune systems like-
- HIV/AIDS
- Solid-organ transplant
- Blood cancer
- Inflammatory diseases or autoimmune diseases i.e. lupus or rheumatoid arthritis
- Stem cell transplant.
Scientific classification
Kingdom: Fungi
Division: Ascomycota
Class: Pneumocystidomycetes
Order: Pneumocystidales
Family: Pneumocystidaceae
Genus: Pneumocystis
Species: P. jirovecii
Binomial name: Pneumocystis jirovecii
Scientists have changed both the classification and the name of this organism since it first appeared in patients with HIV in the 1980s. Pneumocystis jirovecii used to be classified as a protozoan but is now considered a fungus (from 2001). Pneumocystis jirovecii used to be called Pneumocystis carinii. When scientists renamed Pneumocystis carinii to Pneumocystis jirovecii, some people considered using the abbreviation ‘PJP’, but to avoid confusion, Pneumocystis jirovecii pneumonia is still abbreviated ‘PCP.’
Sign and Symptom of Pneumocystis pneumonia
The sign and symptoms of PCP include-
- Fever
- Cough
- exertional dyspnoea
- Chest pain
- Chills
- Fatigue
Extrapulmonary Pneumocystis has also been reported in patients with
advanced HIV disease, particularly in the setting of aerosolized pentamidine prophylaxis. Other organs affected are-
- Orbit
- thyroid,
- skin,
- ears,
- adrenals,
- kidneys,
- bone marrow
- and lymph nodes.
Lab Diagnosis of Pneumocystis pneumonia
Specimen:-
It includes bronchoalveolar lavage (BAL), transbronchial biopsy, sputum, and open lung biopsy. BAL and sputum should always be stained for acid-fast bacillus and other fungi also.
Staining methods for Pneumocystis jiroveci include:
- Gomori’s Methenamine Silver (GMS) stain
- Toluidine Blue O stain
- Giemsa stain
- Fluorescent antibody staining with monoclonal antibody: this is more specific and sometimes more sensitive when a low number of organisms is present in the specimen.
polymerase chain reaction (PCR)
Note: The lifecycle of pneumocystis, is not clearly understood, contains at least two stages. One is the cyst and another trophozoite. The walls of the cyst forms take up the GMS and Toluidine Blue O stain round or cup or typically ‘deflated-ball’-shaped. Trophozoites and cysts are seen in Giemsa stain but cysts having up to eight sporozoites.
Supporting parameters-
- High serum LDH
- Arterial blood gas analysis is very important in the management of cases.
- Chest X-ray
- High-resolution CT scan
Treatment of Pneumocystis pneumonia
The choice of drug used to prevent PCP is trimethoprim/sulfamethoxazole (TMP/SMX), which is also called co-trimoxazole. Its several different brand names are Cotrim, Bactrim, and, Septra.
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