Thirty-two subjects with suspected leprosy lesions were investigated to assess various

modalities of sensibility and sweatfunction and these were correlated with immunological

and histological parameters.

It was found that pain and temperature, mediated by small unmyelinated fibres were

impaired in the early lesions. Impairment of sweat function was seen only when one of the

modalities of sensibility was also affected Antibodies specific to a protein (35 kDa) antigen

and phenolic glycolipid 1 of Mycobacterium leprae were positive in nine and 12 cases

respectively, while 15 of the 31 biopsies revealed the presence of mycobacterial antigens

in these lesions. The implications of these findings are discussed

Balakrishnan, S

Desikan, K V

Karthikeyan, S

Patil, S A

Ramanathan, V D

Ramu, G

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Indian J Lepr Vo1. 68(2) 1996Original ArticleHISTOLOGICAL AND IMMUNOLOGICAL CORRELATES OF SUSPECTEDLEPROSY LESIONSG Ramu, S Kartikeyan, S Balakrishnan, SA Patil, VD Ramanathan, KV DesikanThirty-two subjects with suspected leprosy lesions were investigated to assess variousmodalities of sensibility and sweatfunction and these were correlated with immunologicaland histological parameters.It was found that pain and temperature, mediated by small unmyelinated fibres wereimpaired in the early lesions. Impairment of sweat function was seen only when one of themodalities of sensibility was also affected Antibodies specific to a protein (35 kDa) antigenand phenolic glycolipid 1 of Mycobacterium leprae were positive in nine and 12 casesrespectively, while 15 of the 31 biopsies revealed the presence of mycobacterial antigensin these lesions. The implications of these findings are discussed.INTRODUCTIONEarly detection of leprosy is important in the control of the disease. Treatment of earlylesions avoids deformities and the progress of the disease of infectious types. This is ofobvious importance in a country like India where the majority of cases are paucibacillaryto begin with. Diagnosis is easy in established leprosy since the cardinal signs are readilyelicited (Dharmendra & Chatterji 1978). The criteria for diagnosing leprosy are loss ofsensibility in a patch or enlargement of peripheral nerve trunk or a cutaneous nerve withloss of sensibility in the area of its supply or presence of acid-fast bacilli (AFB) in smearsfrom a suspected patch or an area of skin with altered texture.Lesions are put down as ‘suspected’ when touch sensibility is preserved but othermodalities are impaired in a skin patch and the skin smears are negative for APB or thereare areas of impairment of sensibility in the skin without a patch or nerve thickening, or,there is nerve thickening without loss of sensibility. The microscopic pathology and theimmunological features of established leprosy lesions are well-recognized. However, thehistological and immunological features of the lesions when leprosy is suspected are notknown. In the present study we have attempted to correlate the clinical features of the firsttwo categories of suspected leprosy lesions with some of their immunological, biochemicaland histological parameters.Dr G Ramu, Dr S Kartikeyan, Dr S BalakrishnanSacred Heart Leprosy Centre, Kumbakonam 612 401.Dr SA Patil, Central JALMA Institute for Leprosy, PO Box 31, Taj Ganj, Agra 282 001.Dr VD Ramanathan, Tuberculosis Research Centre, Chetput, Madras 600 031.Dr KV Desikan, Leprosy Histopathology Centre, Mahatma Gandhi Institute of Medical Sciences,Sewagram 442 102.Correspondence and reprint requests: Dr VD Ramanathan.155Vo1. 68(2) 1996PATIENTSThe subjects consisted of 14 male and ten female adults and five male and threefemale children. Twenty-one of these subjects had flat, hypopigmented, ill-defined patchesand ten had areas of numbness in the skin. The commonest site of such numbness was theanterolateral aspect of the thigh. All except two had single lesions.CLINICAL EXAMINATIONThe test for touch sensibility was carried out by using a painting brush no. 4. Adiminution in pain (expressed as less sharp compared to the contralateral side) and loss ofpain were elicited using a 26 G needle. Thermal sensibility was initially tested by using twotest tubes - one with cold water and the other with tepid water. The thermal tester standardizedby Srinivasan and Stumpe (1989) was used later.SEROLOGICAL TESTSImrnunoglobulin M class of antibody to phenolic glycolipid 1 of Mycobacteriumleprae was tested using ELISA as described by Cho and his colleagues (1983). This wasconsidered to be positive if the absorbance value of the sample at a dilution of 1 in 300was greater than 0.250. This value was derived by adding two SD values to the mean of25 control samples. Blood from hospital volunteers served as the control. Antibody to theMy2a epitope of M. leprae was estimated using the serum antibody competition test (SACT)as reported previously (Sinha et al 1983).IMMUNOENZYMATIC STAININGImmunoenzymatic staining of formalin-fixed, paraffin-embedded skin sections wasperformed using anti-BCG antibody (Dakopatts, Denmark) as described by Mshana et al(1982). Immunoglobulin fraction of serum from an unimmunized rabbit served as thenegative control in all the samples. Antigen was seen mainly intracellularly in the macrophagesof the dermal granuloma and occasionally in the Schwann cells of an infiltrated nerve. Thestaining was graded as doubtful (±) and 1+ to 4+ depending on the intensity and extent.Most of the positive specimens showed 1+ to 2+ grading with an occasional 3+.The serological, histological and immunohistological examinations were performedblind without recourse to clinical details.LEPROMIN REACTIONThe Fernandez (at 24 hr) and the Mitsuda (on the 21st day) responses to Dharmendralepromin containing 1x107 Mycobacterium leprae/ ml were measured as described earlier(Sengupta et al 1979).156Indian J Lepr Vo1. 68(2) 1996SWEAT FUNCTION TESTProstigmin 0.02 mg was injected intradermally into the lesion and also into thecontralateral site as control. Assessment of sweat secretion formed was done by the ninhydrinmethod (Rao et al 1987). A filter paper attached to a stamp pad was kept in contact withthe test site for five minutes. The paper was then removed from the stamp pad and sprayedwith 1% ninhydrin in acetone. This gives pink dots wherever there has been sweating. Thecolour was eluted in 75% alcohol containing copper sulphate and expressed as opticaldensity (OD) at 540 nm. Sweat function was considered to be impaired if the OD value ofthe sample from the test site was less than half of the OD value from the control site.RESULTSTouch sensibility was intact at the affected site in all the individuals. Acid-fast bacilliwere not demonstrable either in the smear or in the sections from any of the lesions. Noassociations could be seen when the data were analyzed in relation to age and sex of thesubjects.The patients were divided into three major categories on the basis of the sensorynervous function deficit in the lesions and the results of the various investigations in thedifferent categories are given in Table I. The first category comprised of 20 individuals withimpaired pain and thermal sensibility. The second and third categories consisted of subjectswith lesions where either pain or the thermal sensibility alone was affected.Table I. Results of investigations in 32 patients with ‘suspected’ leprosy lesionsGroups Total no. of Histology Impaired sweat Antigen PGL 1 My2a Mitsudapatients positive function positive +ve +ve positiveI. Impaired A 15 10 10 8 7 3 6pain &thermal B 5 5 * 2 3 3 3 3sensibilityII. Impaired A 3 2 0 2 1 1 2painsensibility B 4 1 1 2 1 -- 1onlyIII. ImpairedthermalsensibilityonlyA 4 1 – – – 2 3B 1 – – – – – 1Group A: Patients with skin patches; Group B: Patients without skin patches but with impaired sensibility.* Three patients showed histological changes suggestive of regressed leprosyHistological evidence of leprosy was seen in 12 of the 20 lesions having both painand thermal sensory impairment. Impairment of sweat function was seen in 12 andmycobacterial antigens could be demonstrated in 11 of these 20 lesions. Antibodies to both157Indian J Lepr Vol. 68(2) 1996PGL and My2a determinant were seen in the sera of four of these individuals. Histologically,three of the lesions showed evidence of regressing leprosy and these individuals had onlysensory involvement without an obvious skin patch. Interestingly, neither antigen nor antibodycould be demonstrated in these subjects. Mitsuda response to Dharmendra lepromin wasobserved in nine of the 20 individuals.In the second group consisting of seven subjects who had only impaired pain sensibilityin the lesions (either analgesia or hypalgesia), histological evidence of leprosy was observedin three of the biopsies. Four of the lesions had evidence of the presence of mycobacterialantigens while the sweat function was affected in only one. Anti M. leprae antibodies wereseen in the sera of four of these individuals while the Mitsuda response was positive in threeof them.The rates of positivity for antigens and antibodies were very low in the five individualswho had lesions with impaired thermal sensibility alone. Histological evidence of leprosywas seen in only one of them. However, four of them showed positive Mitsuda reaction toDharmendra lepromin.DISCUSSIONWhen the cardinal signs of the disease are satisfied, clinical diagnosis of early lesionsof leprosy is possible with a high degree of concordance between clinicians (Neelan et al1982). Thus, leprosy can be diagnosed with certainty when there is loss of touch sensibility,which is mediated by myelinated nerve fibres in a suspected lesion. It is well-known that,in leprosy, unmyelinated fibres which are responsible for pain and thermal sensibility areinvolved earlier than the myelinated fibres (Dastur 1955). Hence, we studied the clinicalfeatures and the immunological and histological correlates of lesions with impairment ofsensory modalities mediated by unmyelinated nerve fibres alone.It was found that a maximal positivity of 50% or more for antigen, antibody, impairedsweat function and histological positivity was observed when both the pain and the thermalsensibilities were affected. This was followed by the group with impaired pain sensibilityalone. The group with only reduced thermal sensibility showed minimal positivity forantigens and antibodies. Similarly, histological evidence of leprosy or impaired sweatfunction could be observed only in one of them. These data together suggest that lesionswith impaired pain and thermal sensibility or those with impaired pain sensibility alone aremore likely to be due to leprosy than the lesions which present with only thermal fibreinvolvement.Further studies are indicated to delineate the relationship between the clearance ofantigen, development of antibody response and delayed hypersensitivity to M. leprae in suchearly cases.Autonomic nerve function is thought to be impaired early in leprosy. However, theresults of sweat function test performed in the present study indicate that this is not invariable.158Indian J Lepr Vol. 68(2) 1996Although the number of patients studied is small, it may be concluded thatdemonstration of antigen in the tissue along with demonstration of antibody in the serumin cases which show impairment of unmyelinated nerve functions is probably diagnostic ofleprosy. However, studies involving larger number of cases using monoclonal antibodiesand/or DNA probes specific to M. leprae are needed to clarify the extent and type ofimpaired functions mediated by unmyelinated nerve fibres in very early leprosy. This wouldthen be of help in laying down clinical criteria to diagnose leprosy in the field withoutrecourse to sophisticated tests involving the demonstration of antigen and/or antibody specificto M. leprae.ACKNOWLEDGEMENTThe authors are indebted to the superintendent, Sacred Heart Leprosy Centre, Kumbakonam for permissionto undertake the study and report it to the Director, CJIL, Agra and the Director, TRC, Madras for permissionto carry out the investigations. The technical assistance of D Krishnan, K Pandi, S Rajamahendran of SHLC andS Jaya Gopinath of TRC is gratefully acknowledged. The authors are grateful to S Seshadri of SHLC for typingthe manuscript.1.2.3.4.5.6.7.8.9.REFERENCESCho SN, Yanaagihara DL, Hunter SW et al 1983. Serological specificity of phenolicglycolipid 1 from Mycobacterium leprae and its use in serodiagnosis of leprosy. Infect Immun41: 1077-1083.Dastur DK 1955. Cutaneous nerves in leprosy. Brain 78: 615-622.Dharmendra, Chatterji SN 1978. The cardinal signs of leprosy. Leprosy Vol 1, ed Dharmendra.Kothari Medical Publishing House, Bombay. p 246.Mshana RN, Belehu A, Stoner GL et al 1982. Demonstration of mycobacterial antigens inleprosy tissues. Int J Lepr 50: 1-10.Neelan PN, Noordeen SK, Ramu G et al 1982. Iner-observer variations in diagnosis andclassification of early lesions of leprosy. Lepr India 54: 485-488.Rao KS, Balakrishnan S, Gommen PK et al 1987. Restoration of plantar sweat secretion in thefeet of leprosy patients. Indian J Lepr 59: 442-449.Sengupta U, Ramu G, Desikan KV 1979. Assessment of Dharmendra antigen. Standardizationof the antigen. Lepr India 51: 316-329.Sinha S, Sengupta U, Ramu G, Ivanyi J 1983. A serological test based on competitive inhibitionof monoclonal antibody binding to My2a determinant of Mycobacterium leprae. Trans Roy SocTrop Med and Hyg 77: 869-871.Srinivasan H, Stumpe B 1989. Leprosy diagnosis: a device for testing the thermal sensibility ofskin lesions in the field. Bull WHO 67: 635-641.159SUBSCRIBERS PLEASE NOTEWe have been receiving a number of copies of the Journal returnedto us undelivered for want of correct address.When a copy is returned to us undelivered we do not mail subsequentcopies until you provide us with your correct mailing address.Please do not fail to include your postal pincode number.Hon. EditorIndian Journal of Leprosy11B/1, First Seaward RoadValmiki NagarMadras - 600 041 (India)

Histological and immunological correlates of suspected leprosy lesions

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