Mycobacterium Leprae - Lab Diagnosis, Nasal, Skin smears, Microscopy, Lepromin skin test
Laboratory diagnosis of Mycobacterium leprae
The laboratory diagnosis of Mycobacterium leprae begins with the collection of samples.
Sample
Samples collected for Mycobacterium leprae diagnosis are
skin scrapings
nose blow
ear lobe skin scrapping nasal swab
biopsy
lymph nodes and affected nerves in lepromatous leprosy.
* In tuberculoid leprosy, the bacilli are not found in these specimens
Skin smears
skin smears for Mycobacterium leprae infection diagnosis are collected from the leprous lesions, such as nodules, thick papules, and areas of infiltration
in cases of patches, the samples are obtained from the edge of the lesion rather than from the center
skin smears are collected from 5-6 different sites including the skin over the ear lobes, buttocks, forehead, cheeks, and chin
Slit and scrap method
in slit and scrap method, the skin is pinched up and raised between the thumb and index finger of the left hand, which squeezes out blood from that part, thereby minimizing bleeding when a cut is made
then with the help of a scalpel, a cut of about 5mm is made on the pinched skin, deep enough into the dermis (2-3 mm) where the bacteria will be found
using a dry piece of cotton wool, blot away any blood which appears at the site of the cur
turn the scalpel blade until it is at a right angle to the cut
using the blunt end of the blade, scrape firmly two or three times along with the edges and bottom of the cut to collect a sample of tissue juice and cells
transfer the sample to a slide. Make a small circular, smear, air dry, heat-fixed, ZN staining and observed under the microscope
while the cur area is covered with a small dressing and instruct the patient to remove the dressing as soon as the cut has healed
Nasal smears
nasal smears from the nasal patches are collected by scraping material from the mucous membrane of the internal nasal septum and spread on the slide and ZN stained.
Microscopy
Smears, for diagnosis of Mycobacterium leprae, are prepared from the sample and are stained by the Ziel-Nelson technique using 5% instead of 20% sulfuric acid for decolonization. The smears after microscopy are graded as below:
1-10 bacilli in 100 field => 1+
1-10 bacilli in 10 field => 2+
1-10 bacilli per field => 3+
1-100 bacilli per field => 4+
10-1000 bacilli per field => 5+
more than 1000 per field => 6+
Bacillary index:
- an expression of the extent of bacterial load
- calculated by counting 6-8 stained smears under the 100 x oil immersion lens
= total no. of pulses in all smears
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no. of smears
Ziel-Nelson staining of Mycobacterium leprae
The procedure followed for Ziel-Nelson staining of Mycobacterium leprae
Prepare the smear from the sample and over the smear filter carbol fuchsin stain
Heat stain until vapor just begins to rise i.e. 60°C. It should not be overheated and the heated stain is allowed to remain on the slide for 10-15 min insuring the stain does not dry on the smear
Wash off the smear with clean water
Decolorize the smear rapidly using acid alcohol
Wash well with water, and cover the smear with malachite green for 1-2 min
Wash off the stain with clear water, wipe the back of the slide clean, and place it on a drying rack for the smear to dry. Protect it from direct sunlight
Examine the smear microscopically
Mycobacterium leprae appears as red solid bacilli, occurring singly or in masses against a green background
Morphological index
percentage of uniformly stained bacilli in tissue is known as the morphological index
uniformity of the bacilli is used as a criterion to differentiate live bacilli from dead bacilli in stain smears
Culture
The culture of Mycobacterium leprae on artificial culture media has not yet been achieved
It is possible, however, to produce growth in armadillo - growth can be achieved in its foot pads
The body temperature of the animal of 30°C which favors the multiplication of the organisms
Lepromin skin test
the Lepromin skin test is used to determine what type of leprosy a person has
It involves the injection of a standardized extract of inactivated leprosy bacillus under the skin
A positive reaction is indicated by an induration of 10mm or above after 48 hours or 5mm or above after 21 days. However, it is not recommended as a primary mode of diagnosis
Two types of reaction are observed after the test:
In an early reaction, there is the formation of induration having >10 mm diameter in 1-2 days which disappears within 4-5 days
the late reaction is characterized by the development of nodules at the site of inoculation after 3-5 weeks of infections
Serodiagnosis
serodiagnosis for detection of Antibody (Ab) against Mycobacterium leprae phenolic glycolipid Ag is carried out to screen the presence of Mycobacterium leprae
ELISA and latex agglutination tests are used to detect serum Ab.
Molecular diagnosis
A molecular diagnosis such as PCR for identifying DNA that encodes 65 and 18 kDa Mycobacterium leprae proteins
Physical examination
Physical examination for Mycobacterium leprae is done by following procedures:
evaluation of skin lesions
careful sensory and motor examination
palpation of peripheral nerves for pain or enlargement. Particular attention should be paid to the following locations:
- elbows: Ulnar nerve
- wrist: superficial radial cutaneous and median nerves
- popliteal fossa – common peroneal nerve
- neck – great auricular nerve