Specimens:
- It includes nasal mucosa, skin lesions, ear lobules.
- Occasionally, lymph nodes and affected nerves.
Method of collecting of skin smears:
Slit and scrap method:
- Ask the patient to sit with his or her back to the table on which the equipment for taking the smears is placed.
- Decide the sites from where the smears are to be collected and mark these areas on the outline drawing of the body in the patient serum.
- Take a completely clean slide (frosted ended) and using a lead pencil label the slide with the date and the patients name and numbers.
- Fit a new scalpel bladed in its scalpel holders, sterile the blade by wiping it carefully with a piece of absorbent cotton wool soaked in 70 % v/v ethanol and flaming it for 2-3 seconds in the flame of a spirit lamp.
- Allow the blade to cool.
- Cleanse the area from where the smear is to be taken using a cotton wool swab soaked in 70 % v/v ethanol.
- When dry hold a fold of the skin tightly between the thumb and index finger until it becomes pale due to loss of blood.
- Using the sterile blade, make a small cut through the skin surface, 5 mm long and deep enough into the dermis (2-3mm) where the bacteria will be found, continue to hold the skin tightly.
- Using dry cotton, blot away any blood which appears at the site of the cut.
- Trim the scalpel blade until it is at a right angle to the cut. Using the blunt edge of the blade, scrap firmly two or three times along the edges and bottom of the cut.
- Make a small circular smear of the tissue juice and cells, covering evenly the area of the slide about 5-7 mm in diameter.
- Stain smear by ZN technique.
Collection of nasal smears:
- Request the patient to sit facing the light with his/her head bent backwards so that the nasal cavities are visible.
- Using an absorbent cotton wool swab insert the swab in to one of the nasal cavities. Relate and rub the swab several times against the upper part of the nasal sputum.
- Make an evenly spread smears of the material on a labelled slide covering an area about 7 mm in diameter.
- Allow the smear to air dry
- Stain by ZN technique
1. Microscopy:
- Ziehl- Neelsen method:
- Gently heat-fix the smears
- Cover the smears with filtered carbolfuchsin stain
- Heat the stain until vapor just begins to rise i.e. 60 degree Celsius
- Wash the stain
- Decolorize the smears rapidly with 1 % v/v acid alcohol for 10 second for skin smears and 15 sec for nasal smears
- Wash well with water
- Cover the slide with malachite green for 1-2 min
- Wash off the stain with water
- Allow the slide to air dry.
- Examine under microscope.
- Observations:
- M. leprae– red solid bacilli or beaded, fragmented, granular forms occurring singly or masses as globi.
- Macro phages cell: green
- Background material: green
- Reporting of M. leprae smears:
- If globi are seen, report their number as few, moderate or many.
- The concentration of organisms in each smear is estimated and reported as a Bacterial Index (BI).
- BI is defined as number of viable bacilli in a lesion which is assessed from stained smear by oil immersion lens (*100) of microscope.
- It is calculated by totaling the number of pluses (+S) scored in all smears divided by the number of smears.
- For calculating BI< a minimum of four skin lesions or nasal swab and both the ear lobes have to be examined.
- BI helps to determine the progress and treatment of a patient.
- BI is affected by the thickness of the film, depth of the skin incision and thoroughness of the scrap.
- Internationally agreed bacteriological index ranges from 1 to 6 + as follows:
- 1-10 bacilli in 100 fields= 1+
- 1-10 bacilli in 10 fields=2+
- 1-10 bacilli/ field = 3+
- 10-100 bacilli/ field = 4+
- 100-1000 bacilli/field = 5+
- >1000 bacilli/ field = 6+
- If after examining 100 fields no bacteria are found before reporting the smear as ‘BI’= 0, examine several more areas of the smear.
- Average BI for patient = 2.4
- The morphological Index (MI) is the percentage of uniformly stained bacilli out of the total number of bacilli counted. It is useful in assessing the prognosis and response to treatment.
2. Lepromin skin test:
- Lepromin test is a non-specific skin test which can be helpful in classifying leprosy and assessing the future course of the disease.
- One of its main values is in confirming lepromatous leprosy (LL) when it is always negative. The test was first described by Mitsuda in 1919.
- The antigen used is the lepromin antigen, which is a suspension of killed M. leprae. Obtained from infected human or armadillo tissue.
- Leprosins are another type of skin testing reagent. These are ultrasonicates of tissue-free bacilli extracted from lesions.
Procedure of Lepromin test:
- 0.1ml of lepromin (containing 40 millions dead lepra bacilli) is injected intradermally into the forearm.
- The response to the intradermal injection of lepromin is typically biphasic consisting of two separate events.
- The first or Fernandez reaction, consists of erythema and induration developing in 24-48 hours. It is about 10-30 mm diameter and disappear within 3-5 days.
- The second Mitsuda reaction is characterized by erythematous nodular lesions of 3-5 mm diameter that develops at the site incubation in 3-5 weeks.
- Positive reaction indicates resistance to leprosy.
- The test is negative in lepromatous leprosy but positive in tuberculoid leprosy.
- Lepromin test is not used to diagnose leprosy
3. Culture:
- M. leprae has not yet been cultured invitro either in bacteriological media or tissue culture.
- Attempts have been made to culture lepra bacilli in experimental animal.
- Footpads of mice:
- Inoculation of ground tissue or nasal scrapings from lepromatous leprosy containing lepra bacilli intradermally into foot pad of mouse kept a low temperature (20 degree Celsius) results granulomatous lesions at the site of injection in 1-6 month of time.
- Nine banded Armadillo:
- In armadillo (Dasypus novemcinctus), generalized infection occurs with extensive multiplication of the bacilli and production of lesions typical of lepromatous leprosy.
- Natural infections by mycobacterium spp. resembling M. leprae have also been observed in some wild armadillo in Texas and Mexico.
- Other experimental animals include slender loris, Indian pangolin and Korean chipmunks.
4. Biopsy:
- Skin and nerve biopsy are useful method to diagnose leprosy.
- Skin biopsy is collected from active edge of the patches and nerve biopsy from the thickened nerve for histological confirmation of tuberculoid leprosy when AFB cannot be demonstrated in direct smear.
- Tuberculoid leprosy shows infiltration of lymphocytes around the center of epithelial cells, presence of langhans giant cells, few or no AFB.
- Lepromatous leprosy shows predominantly foamy macrophages with few lymphocytes and no giant cell.
- The other methods that are also useful in detecting viable lepra bacilli include:
- Fluorescent diacetate-ethidium bromide (FDA-EB) staining
- Laser microscope mass analysis (LAMMA), bioluminescent technology
- Macrophages based assays.
5. Serodiagnosis:
- Serodiagnosis of leprosy involves detection of antibiotic to M. leprae specific PGL-1 antigens.
- ELISA, latex agglutination test, Mycobacterium leprae particle agglutination (MLPA) are used to detect serum antibodies.
6. Molecular diagnosis:
- PCR for identifying DNA that encodes 65 and 18 kDa M. leprae proteins and repetitive sequences of M. leprae is being used to detect and identify M. leprae in clinical specimens.
Treatment:
- Dapsone was the first effective chemotherapeutic agent against leprosy.
- However due to development of drug resistance, WHO has recommended multidrug therapy (MDT) for all leprosy cases based on dapsone, rifampicin and clofazimine.
Prevention and control:
- Early diagnosis
- Treatment of leprosy
- Surveillance of contacts
- Health education
- Vaccines- BCG
- Chemoprophylaxis