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ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 4  |  Page : 213-216

Role of S100 immunostaining in demonstrating neural granulomas in paucibacillary leprosy


1 Department of Pathology, IMS and SUM Hospital, Bhubaneswar, Odisha, India
2 Department of Pathology, ESI Hospital, Joka, West Bengal, India

Date of Submission27-Apr-2020
Date of Acceptance29-Apr-2020
Date of Web Publication6-Jan-2021

Correspondence Address:
Dr. Debasmita Das
Department of Pathology, IMS and SUM Hospital, Bhubaneswar, Odisha
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_64_20

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  Abstract 


Background: Challenges encountered in diagnosing paucibacillary leprosy are difficulty in visualizing the nerve twigs in H&E section due to small size or fragmentation and paucity of organisms in Wade-fite stain.
Aims: This study aims to test the role of S100 immunostain in demonstration of neural granuloma, the hallmark in paucibacillary leprosy, compare morphology of S100 staining of nerves inside granuloma in different types of leprosy and distinguish this pattern from non-leprosy cutaneous granulomas.
Methods: Sixty cases of diagnosed paucibacillary leprosy were studied in a span of one and half year (January 2018 to June 2019). Ten cases of non-lepromatous cutaneous granulomas and five cases of normal skin histology were also studied and included as control. H&E stain along with S100 immunostaining was done for all the cases. The H&E stained slides were used for studying the histopathological features and the S-100 was used for identifying the different dermal nerve patterns.
Results: The most common pattern seen in case of borderline tuberculoid was fragmented and infiltrated whereas most common pattern seen in case of indeterminate was intact and infiltrated. Reduced, fragmented and infiltrated was most commonly seen in case of tuberculoid leprosy. Conclusion: Immunohistochemistry with S-100 has shown to be an effective adjuvant to histopathology in the diagnosis of different types of paucibacillary leprosy with bacterial index zero and to differentiate them from non-lepromatous granulomatous condition.

Keywords: Bacillary index, cutaneous granuloma, paucibacillary Hansen's disease, S100 immunostaining


How to cite this article:
Mohapatra D, Das D, Chakraborty K. Role of S100 immunostaining in demonstrating neural granulomas in paucibacillary leprosy. J NTR Univ Health Sci 2020;9:213-6

How to cite this URL:
Mohapatra D, Das D, Chakraborty K. Role of S100 immunostaining in demonstrating neural granulomas in paucibacillary leprosy. J NTR Univ Health Sci [serial online] 2020 [cited 2021 Jan 18];9:213-6. Available from: https://www.jdrntruhs.org/text.asp?2020/9/4/213/306127




  Introduction Top


Leprosy is one of the oldest known diseases to mankind and India holds the unenviable position of its origin. According to the latest data by the National Leprosy Eradication Programme (NLEP), the incidence of new case detection is 9.27/100,000 population in the year 2017–18.[1] Leprosy is seen as a social stigma that brings anxiety in the minds of people.[2] Thus, utmost care should be taken in the diagnosis of leprosy under Odisha Health and Family welfare with a target to eradicate leprosy by March 2020.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which is seen presenting itself in different clinicopathological forms. There are varieties of differential diagnostic possibilities of skin granulomas to histopathologists which include paucibacillary leprosy and non-leprosy granulomatous inflammation such as tuberculosis, sarcoidosis, fungal infections, and chromoblastomycosis.[3],[4],[5]

Histopathology is a key to the diagnosis of leprosy. It is usually not difficult in case of the lepromatous spectrum as the bacillary index is high but often possesses difficulty in the case of tuberculoid and indeterminate forms which are paucibacillary type. Clinically and morphologically, these overlap with other non-lepromatous granulomatous inflammation. Even the Fite-Faraco technique and culture are often unrewarding and cumbersome where the bacterial index is zero.

S100 is an immunohistochemical marker of Schwann cells of peripheral nerves, it can be used as an auxiliary tool to identify nerve destruction and distinguish patterns of paucibacillary leprosy which is difficult to diagnose in H and E stained sections.

Thus, the aim of the study is:

  1. to know the role of S100 immunostain pattern in demonstrating neural granulomas in skin biopsies of paucibacillary leprosy (tuberculoid [TT], borderline tuberculoid [BT], and indeterminate type),
  2. to compare the morphology of S100 staining of nerves inside granulomas among clinico-histologically defined different categories of leprosy, and
  3. to distinguish it from other non-lepromatous granulomatous inflammation based on the S100 staining pattern.



  Materials and Methods Top


The study was conducted in the Department of Pathology, IMS and SUM hospital from January 2018 to June 2019. Sixty histologically diagnosed cases of paucibacillary leprosy and 10 cases of cutaneous non-lepromatous granuloma were studied which were taken as controls. The following cases were excluded from the study: Lepromatous cases, type I and II lepra reactions, patients on medication for leprosy, and relapse after inadequate treatment.

Biopsies were adequately fixed in formalin and processed to prepare paraffin blocks. H and E and Wade-Fite stain were done on all the skin biopsies of interest. Immunohistochemistry for S100 protein was done on 3 μm thick tissue sections. For each staining batch, appropriate positive control was kept.

While interpreting S100 positivity, morphology was used to distinguish nerve fragments from other cells such as macrophages, sweat gland ducts, and Langerhans cells, which also show S100 positivity. The presence of nuclear and cytoplasmic staining of nerve twigs was taken as the criterion for the visualization of nerves on S100 immunostaining. One intact nerve bundle with continuous fibrillar structure was considered adequate in the control slides.

Biopsies were screened for the following patterns:

  1. Intact: Dark staining, large fibrillar structures in a wavy pattern
  2. Intact and infiltrated: dark staining, large fibrillar structures in a wavy pattern with inflammatory cells inside
  3. Fragmented and infiltrated: The discontinuous staining pattern with small nerve twigs separated and infiltrated by a dense inflammatory infiltrate
  4. Reduced, fragmented, and infiltrated: Very few nerve twigs scattered in a background of dense inflammation.


Results obtained from leprosy and non-lepromatous granulomatous inflammation were compared and the significance of the difference was calculated by using the Chi-square test. Applicability of the S100 immunostaining to differentiate between fragmented/destructed nerves in leprosy from normal uninvolved nerves by using Fisher's exact test.

Ethical Clearance

Ethical approval for this study (Ethical Committee ECR/627/Inst/OR/2014/PR-2) was provided by the institutional Ethical Committee, institute of Medical Sciences (IMS) and SUM Hospital in December 2017.


  Results Top


Out of 60 cases of paucibacillary leprosy studied, 6 cases were tuberculoid leprosy, 40 cases were borderline tuberculoid leprosy and 10 cases were indeterminate leprosy. Only four cases were mid-borderline.

The most common patterns observed were reduced, fragmented and infiltrated in case of TT leprosy [Figure 1], fragmented and infiltrated in case of BT leprosy [Figure 2] and intact and infiltrated in case of indeterminate (I) leprosy [Figure 3]. 50% mid-borderline leprosy showed infiltrated and intact and 50% were fragmented and infiltrated [Table 1].
Figure 1: Microphotograph of tuberculoid leprosy shows well-formed granulomas. (H and E, 400×) and reduced, fragmented, and infiltrated nerve fibers by granulomas. (IHC, S100, 400×)

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Figure 2: Microphotograph of borderline tuberculoid shows granulomas. (H and E, 400×); fragmented and infiltrated nerve fibers by granulomas. (IHC, S100, 400×)

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Figure 3: Microphotograph of Indeterminate leprosy shows periappendegeal inflammatory infiltrate (H and E, 100×); with intact and infiltrated nerve fibers by inflammatory cells. (IHC, S100, 400×)

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Table 1: Morphology of Nerve Fragments Observed on S100 Immunostaining

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Eight cases out of 10 cutaneous non-leprosy granulomas along with normal skin biopsy showed intact nerve bundles. Applicability of S100 immunostaining to differentiate between fragmented/destructed nerves in leprosy from normal uninvolved nerves was found to be significant. (P value using Fisher's exact test = <0.001).

Cutaneous granulomatous (non-leprosy) cases which included four cases of tuberculosis verrucosa cutis [Figure 4], three cases of lupus vulgaris, and one case of erythema nodosum and 2 cases of sarcoidosis were studied. Here, the most common pattern of staining for S100 seen was intact without inflammatory change. The probability to differentiate between leprosy and non-lepromatous granulomas based on the pattern of staining of the nerve within granuloma on S100 immunostaining was statistically significant (P < 0.001, x2 = 70, df = 3) [Table 2].
Figure 4: Microphotograph of tuberculosis verrucosa cutis shows periappendegeal acute and chronic inflammatory infiltrate (H and E, 400×); shows intact nerve bundle (IHC S100, 400×)

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Table 2: Probability of S100 to Differentiate Between Leprosy and Non-Leprosy Granuloma

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  Discussion Top


Leprosy is a neurotropic disease caused by M. leprae. The disease is endemic in many tropical and subtropical countries especially in the Eastern part of India.[6],[7],[8] The diagnosis of leprosy brings anxiety in the minds of people not only for the deformities associated with it but also a social stigma.

However, it brings many challenges to the pathologist. The histologic diagnosis in tuberculoid (BT and TT) and indeterminate type is difficult and frequently not possible because of difficulty in detecting lepra bacilli with Wade-Fite stain. Granulomatous reaction with the destruction of neurovascular bundles are considered to be an important criterion for diagnosing tuberculoid and indeterminate leprosy.

S100 is a sensitive and reliable marker for nerve destruction. Fleury and Bacchi in their study were able to visualize peripheral nerves in tuberculoid granuloma by staining for S100.[9] In the present study, all the 60 cases of paucibacillary leprosy showed neural staining inside the granulomas by S100. Ismail EA demonstrated increased nerve bundles by S100 immunostain in leprosy than in non-Hansen's granulomas. Nerve bundles were much better visualized using S100 than H and E stain.[10]

Similar results were reported by Gupta et al.[11] and Mohanraj and Srinivasan.[12] In the above two studies, there was no uniformity in the pattern definition set. The destruction of nerves by inflammation is significant within the granulomas and not outside. In the present study, only tuberculoid (BT and TT) and indeterminate leprosy were taken into consideration and nerve bundles either intact or fragmented found inside the granulomas were taken into account. Thus the patterns studied in our study were intact, intact and infiltrated, fragmented and infiltrated and reduced, fragmented, and infiltrated. The difference in staining patterns of nerve fragments by S100 provides a clue about the spectrum to which a case of leprosy belongs similar to Shenoy N, et al.[13]

Thomas et al. compared the morphology of nerve bundles based on the pattern of the S100 staining in cases of leprosy and non-lepromatous granulomatous inflammation.[14] They had taken three patterns into consideration, that is, intact, fragmented, and absent. The intact nerve had been taken as controls found in non-lepromatous conditions. In the present study, there were ten non-Hansen's granulomatous inflammation cases. All of these cases showed an intact pattern without inflammation. Hence, the probability to differentiate leprosy and non-lepromatous granulomas in terms of the S100 staining pattern was found to be statistically significant.

All of the above studies are correlating with our result except Singh et al.[4] who reported that the most reliable criteria for diagnosing leprosy are the complete absence of nerve twigs.


  Conclusion Top


S100 can be used as an auxiliary tool for identifying damaged nerve bundles in the case of paucibacillary leprosy which is not visualized by H and E stain. It can also be helpful in differentiating leprosy from non-lepromatous granulomas. The different staining patterns of S100 may provide a clue about the spectrum to which a case of leprosy belongs, especially the mid-borderline and indeterminate type of leprosy which is very difficult to diagnose in H and E stained section, thus helping these therapeutically responsive entities.

Financial support and sponsorship

Nil.

Conflicts of interest?

There are no conflicts of interest.



 
  References Top

1.
National Leprosy Elimination Programme: Training manual for medical officers 2019. https://cltri.gov.in/MO%20Training%20Manual.pdf. [Last accessed on 2020 Jan 10].  Back to cited text no. 1
    
2.
Gelber RH. Leprosy. In: Longo DL, Fauci AS, Kasper DL, Hauser SL, Jameson JL, Loscalzo J, et al. editors. Harrison's Principles of Internal Medicine. 18th ed. New York: McGraw Hill; 2012. p. 1359-67.  Back to cited text no. 2
    
3.
Job CK. Pathology of leprosy. In: Hastings RC, Opromolla DV, editors. Leprosy. 2nd ed. Edinburgh: Churchill Livingstone; 1994. p. 193-234.  Back to cited text no. 3
    
4.
Singh N, Arora VK, Ramam M, Tickoo SK, Bhatia A. An evaluation of the S-100 stain in the histological diagnosis of tuberculoid Leprosy and other granulomatous dermatoses. Int J Lepr Other Mycobact Dis 1994;62:263-7.  Back to cited text no. 4
    
5.
Khan AR. S-100 protein in the diagnosis of tuberculoid/borderline tuberculoid leprosy. Ann Saudi Med 1998;18:305-7.  Back to cited text no. 5
    
6.
Lucas S. Bacterial diseases. In: Elder DE, editor. Lever's Histopathology of the Skin. 10th ed. Wolters Kluwer; 2009. p. 558-66.  Back to cited text no. 6
    
7.
Noordeen SK. Eliminating leprosy as a public health problem. Int J Lepr 2004;63:559.  Back to cited text no. 7
    
8.
Briton WJ, Lockwood DNJ. Leprosy. Lancet 2004;363:1201-19.  Back to cited text no. 8
    
9.
Fleury RN, Bacchi CE. S-100 protein and immunoperoxidase technique as an aid in the histopathologic diagnosis of leprosy. Int J Lepr Other Mycobact Dis 1987;55:338-44.  Back to cited text no. 9
    
10.
Ismail EA. Comparison of leprosy and non-leprosy granulomas by using S-100 protein. J Med Res Inst 2007;28:181-4.  Back to cited text no. 10
    
11.
Gupta SK, Nigam S, Mandal AK, Kumar V. S-100 as a useful auxiliary diagnostic aid in tuberculoid leprosy. J Cutan Pathol 2006;33:482-6.  Back to cited text no. 11
    
12.
Mohanraj A, Srinivasan S. Role of S-100 immunostaining in demonstration of nerve changes and quantification of dendritic cells in leprosy. J Clin Diagn Res 2014;8:38-40.  Back to cited text no. 12
    
13.
Shenoy N, Nair NG. Study of S100 immunostaining in demonstrating neural granulomas in paucibacillary leprosy. Indian J Dermatol 2018;63:215-9.  Back to cited text no. 13
[PUBMED]  [Full text]  
14.
Thomas MM, Jacob M, Chandi SM, George S, Pulimood S, Jeyaseelan L, et al. Role of S-100 staining in differentiating leprosy from other granulomatous diseases of the skin. Int J Lepr Other Mycobact Dis 1999;67:1-5.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

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