Laboratory Diagnosis of Cutaneous Leishmaniasis
- Cutaneous leishmaniasis in Ethiopia is caused by the following Leishmania
species: L. tropica, L. major, and L.aethiopica
Laboratory diagnosis is based on detecting amastigotes in smears taken from
infected ulcers or nodules.
Collection and Examination of Slit Skin Smears for Amastigotes
Material for examination should be taken from the inflamed raised swollen
edge of an ulcer or nodule. Its base or center, which usually contains only
necrotic tissue should be taken to avoided because it can contaminate the
specimen with blood and is low yield for amastigotes.
Note
: Secondary bacterial contamination makes it difficult to find parasites
and therefore if bacterial infection is present, delay examination for
leishmania amastigotes until antimicrobial treatment has been completed and
the bacterial infection has cleared.
Method
1. Cleanse the area with a swab soaked in 70% v/v alcohol. Allow drying
completely.
2. Firmly squeeze the edge of the lesion between the finger and thumb to
drain the area of blood (protective rubber glove should be worn)
3. Using a sterile scalpel blade, make a small cut in to the dermis and
blot any blood. Scrape the cut surface in an out ward direction to obtain
tissue juice and cells.
4. Spread the material on a clean slide using a circular motion and working
outwards to avoid damaging parasites in those parts of the smear that have
started to dry. The smear must be thinly spread and not left as thick ‘dab’
smear. Parasites are difficult to find in thick smears.
5. When dry, fix the smear by covering it with a few drops of absolute
methanol – Fix for 2-3 minutes and stain the smear using the Giemsa
technique.
Giemsa staining techniques
Reagents required: -
• Giemsa stain
• Buffered water, PH 7-1 – 7.2 or
• Buffered saline, PH 7.1- 7.2
Method
A. Immediately before use, dilute the Giemsa stain to make 10% solution.
This solution requires minutes staining time;
Preparation of 10% solution
: Measure 45 ml of buffered water
Ph 7.1 - 7.2 in a 50ml cylinder- Add 5 ml of Giemsa stain (to 50 ml mark)
and mix gently
B. Place the slides in a shallow tray, supported on two rods, in a coplin
jar, or in a staining rack for immersion in a staining trough
C.Pour the diluted stain into the shallow tray, Coplin jar, or stain
thoroughly and stain for 10 minutes
D. Wash the stain from the staining container using clean water
B. Wipe back of the slide clean and place it in a draining rack to air
–dry.
6. When the smear is dry, spread a drop of immersion oil on it and examine
first with the 10 x and 40 x objectives to detect macrophages which may
contain amastigotes (the parasites can also be found outside macrophages)
use the 100-X oil immersion objective to identify the amastigotes.
Morphological characteristic of amastigotes
- Amastigotes are – small round to oval bodies measuring 2-4 μm
Can be seen in groups inside blood monocycles (less commonly in neutrophils), in macrophages in aspirates or skin smears, or lying free between cells
• The nucleus and rod-shaped kinetoplast in each amastigotes stain dark reddish mauve
• The cytoplasm stains pale and is often difficult to see when amastigotes are clustered in a group.
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