Lepromin test & Dharmendra antigen

Skin test with lepromin is the only in vivo immunological skin test used as an aid to the classification and prognosis of leprosy

Lepromin test:

• The Iepromin test was first described by Mitsuda in 1919
• this test used intradermal injection of a lepromin ( Dharmendra or Mitsuda antigen), to classify the stage of leprosy based on the lepromin reaction (Fernandez or Mitsuda reaction); it differentiates tuberculoid leprosy, in which there is a positive delayed reaction at the injection site, from lepromatous leprosy, in which there is no reaction (a negative test result) despite the active malignant Mycobacterium leprae infection

Two types of antigens are in use—Mitsuda and Dharmendra lepromins

The Lepromin lest could be compared lo the Tuberculin lest since it is also a skin test intended to assess the delayed types of hyper sensitivity (DTH) due to a specific infection. However, there are certain basic differences in the nature of antigen between Tuberculin and Lepromin.

1. Tuberculin is a culture filtrate of tubercle bacilli. Since leprosy bacilli cannot be cultured, lepromin is a suspension of whole or partially defattened leprosy bacilli killed generally by heat,
2. Secondly, since the only source of leprosy bacilli is the human or armadillo host, contamination by the host tissue is unavoidable despite all the attempts to purify the organisms from the tissue contamination

The original Mitsuda antigen — the crude or integral lepromin — is prepared by grinding the tissues rich in bacilli in 0.5% carbol saline, the larger particles being removed by filtration. The lepromin is standardized by the weight of the whole tissue. It is obvious that lepromin prepared by this method would contain a considerable proportion of human tissues, Fernandez and Castro purified the integral lepromin by differential centrifugation, but the yield of bacilli by this method was not high.

The lepromin extensively used in India is the one prepared according to the method described by Dharmendra (1967) in which the organisms arc extracted from the tissues with chloroform. The lepromin thus prepared is comparatively free of host (issues, but the bacilli are partially defatted on account of the chloroform used). The standardization of Dharmendra lepromin is also by weight, 10 mg of dried bacillary powder being dissolved in 100 ml carbol saline

• It is chloroform-ether extracted suspension of Mycobacterium leprae; used to produce the lepromin test.
• Dharmendra antigen is a suspension of de­fatted leprosy bacilli first reported by Dharmendra in 1941-1942. But it was fur­ther standardised by bacterial count by Sengupta et al 1979. This antigen evokes an early as well as late reaction with an intrad­ermal dose of 0.1ml.

Method

0.1 ml of lepromin is injected intradermally usually on the fore-arm. Positive reaction is observed at two different times—48 hours and 21 days after the injection.

Early reaction (Fernandez) :The 48 hour reaction or the early reaction is seen as a wheal with slight edema and-induration. The diameter of the wheal and induration is measured by a pair of calipers. The reaction is a manifestation of the classical delayed type hypersensitivity and is weak with Mitsuda lepromin. Induration of 5 mm and above with erythema of 10 mm or more was considered as positive.

late reaction (Mitsuda) :The 21 day reaction or the late reaction is seen as a local induration of skin producing a nodule like firm swelling. In a strong reaction the nodule often ulcerates. The diameter of the induration is measured. The reaction is strong with Mitsuda lepromin but weak with Dharmendra preparation. On histological examination, the nodule shows collections of numerous lymphocytes with epithelioid cells and occasionally giant cells. The picture corresponds to that of tuberculoid type of leprosy

Lepromin reaction in cases of leprosy

The positivity of Lepromin test runs parallel with the Cell Mediated Immunity. Therefore, it is strongly positive in cases of Tuberculoid leprosy and negative in lepromatous leprosy. On the Borderline and Intermediate types it has an intermediate response, and various according to the C.M.I.

The present concept of leprosy is that of an immunological spectrum—spreading from

• highly resistant Tuberculoid (TT)
• through the Borderline group :The Borderline group can further be divided into 3 types —
• the Borderline towards the Tuberculoid (BT),
• the truly Borderline (BB) and
• the Borderline towards the Lepromatous (BL).
• practically non-resistant Lepromatous (LL),

The above 5 groups in addition to a larger number of intermediate forms, show gradual gradation in the resistance from the Tuberculoid to the Lepromatous end.

The lepromin test shows a corresponding fall from the strongest reaction in Tuberculoid leprosy to the Negative response in lepromatous leprosy with variable degrees of positivity in the different types of Borderline cases.

It is obvious from the results of the lepromin test that the Tuberculoid (TT) type shows the highest host resistance, while the lepromatous type is “anergic” to the skin test and the Borderline group show varying degrees of positivity depending on their position in the immunological spectrum.

The anergic state of lepromatous leprosy is the most interesting phenomenon in leprosy which is not seen in other diseases.

In tuberculosis at any rate, there is no condition parallel to lepromatous leprosy. There is thus perhaps an immune paralysis in lepromatous leprosy which accounts for the extremely large number of bacilli much greater than the number of bacilli found in the tissues of even moribund cases of tuberculosis. Thus the anergy in lepromatous leprosy seems to be a distinctive feature.

Significance of Lepromin test in cases of Leprosy

• The most important significance of lepromin test is in its application in classification of the disease.
• While in a good number of cases, diagnosis is possible by clinical examination alone, a proper classification of the disease requires clinical and histological examination, assessment of bacterial load by skin smears, as well as by lepromin reaction.
• The lepromin reaction in different types of leprosy is indicated below:

Tuberculoid type (TT)	2+to3+
Maculo anaesthetic type (MA)	1 + to 2+
Indeterminate type (I)	Neg. ± or +
Borderline type (BT)	1 + to 2 +
(BB)	±or +
(BL)	Neg. or ±
Lepromatous type (LL)	Neg.

It is obvious that Lepromin cannot be used as a diagnostic test since the very malignant form of the disease, namely the lepromatous type as well as some of the intermediate groups are negative.

Even positive test has no significance since the test indicates a delayed hypersensitivity reaction to an exposure to M. leprae. As such it could be expected to be positive in a good proportion of persons living in endemic areas and also in contacts of leprosy patients who might have a sub-clinical infection without manifestation of the disease.

While it is not a diagnostic test, lepromin helps in assessing the prognosis of a case.

• Thus, a patient of the Indeterminate type of leprosy with negative lepromin test has a grave prognosis since it is likely to turn into the lepromatous variety.
• Similarly a patient with 2+lepromin test belonging to the Maculo-anaesthetic type or Borderline (BT) type has a favourable prognosis