histopathology of skin lesions of leprosy before and after ......macrophage granuloma was taken as...

Histopathology of skin lesions of leprosy before and

after fixed duration treatment

SARITA SASIDHARANPILLAI*,

APARNA GOVINDAN*, NAJEEBA RIYAZ*,

MANIKOTH PAYYANADAN BINITHA*, SATHI

PUTHEN PARAMBATH*, ANZA KHADER*,

PABIN PAVITHRAN**, DEEPTHI NALINI SURESHAN*,

NIRMAL CHANDRASEKHAR*** &

NEETHU HARIDAS*

*Government Medical College, Kozhikode, Kerala, India

**Nirmala Hospital, Kozhikode, Kerala, India

***Community Health Centre, Irikkur, Kannur, Kerala, India

Accepted for publication 15 August 2016

Summary

Objectives: To study and compare the histopathological features of skin lesions of

leprosy before and after fixed duration treatment.

Design: Prospective study. The first 30 newly diagnosed leprosy patients from July

2012 who successfully completed fixed duration treatment from our tertiary care

institution, and who were willing for a post-treatment skin biopsy were included in

this study after obtaining written informed consent. Only those who underwent a

pre-treatment biopsy were enrolled in the study. Histological features of pre- and

post-treatment biopsies of the skin lesions of leprosy were studied and compared.

Results: The patients who showed an increase or only a slight reduction in

granulomas post-treatment compared to the pre-treatment status had clinical and/or

histological evidence of lepra reaction at the completion of treatment or had lepra

reaction during FDT which was not managed with steroids. Two patients whose pre-

treatment biopsy revealed only inflammatory infiltrate manifested granulomas in the

post-treatment biopsy. 18/30 developed dermal fibrosis after treatment, which was

more common in those with considerable post-treatment reduction in inflammation.

Five patients showed post-treatment restoration of the reduced basement membrane

pigmentation that was noted in the pre-treatment biopsy.

Conclusions: Persistence or increase in granulomas after treatment was not a bad

prognostic sign. More prospective studies with a larger sample size analyzing the

histological resolution achieved by FDT may improve our knowledge of leprosy.

Keywords: Leprosy, Fixed duration treatment, Histopathology

Correspondence to: Sarita Sasidharanpillai, Government Medical College, Kozhikode, Kerala, India (e-mail:[email protected])

Lepr Rev (2017) 88, 142–153

142 0305-7518/17/064053+12 $1.00 q Lepra

Introduction

Though more than a century has passed since the identification of Mycobacterium leprae as

the causative organism for leprosy, several aspects of this ancient disease remain unclear to

this date. Multidrug treatment has achieved elimination of leprosy as a public health problem

and now the focus has shifted to handling the challenges after treatment.

Effective management of relapse in treated patients is an important step in consolidating

the gains achieved by fixed duration treatment (FDT). Occasionally it becomes difficult to

distinguish relapse from late lepra reactions (lepra reactions occurring after completion of

FDT). Scarcity of data on the histological resolution that could be expected at the end of FDT

has added to this confusion.

Poricha et al. in their study of the histology of nerve lesions of treated leprosy cases

observed that a compact granuloma with a dense collar of lymphocytes around a few

epithelioid cells and without giant cells is more in favour of a resolving granuloma.1 By this

criterion, the presence of giant cells is suggestive of activity. Many, including the present

authors, have observed the appearance of giant cells in lepra reactions.2

Trindade et al. observed that indeterminate leprosy (IL) patients manifested typical

leprosy granulomas post-treatment. The authors suggested two explanations for this - either it

shows that the natural course of disease cannot be altered in at least some of the patients or it

supports the fact that bactericidal activity of drugs results in bacillary killing leading to

improvement in immune status of the individual against Mycobacterium leprae and evoking a

granulomatous reaction. In the same paper, it was opined that it is often difficult to distinguish

histologically between granulomatous reactivation due to lepra reaction and leprosy relapse,

and that bacterioscopy alone is useful in such situations.3

It has been documented that treated leprosy lesions show an increase in epidermal

basement membrane pigmentation and morphoea-like changes in the dermis (sclerotic dermis

with paucity of adenexal structures and inflammatory infiltrate).4 A better delineation of the

histological features of treated leprosy lesions may enable us to understand more about the

complex immunological changes in leprosy.

So we considered it worthwhile to study the histopathological findings at the end of FDT

for PB, smear negative and smear positive MB leprosy and compare it with the pre-treatment

status.

Materials and Methods

STUDY DESIGN: PROSPECTIVE STUDY

Written informed consent was collected from each study subject using a standard consent

form. The study adhered to the International Guidelines for Biomedical Research Involving

Human Subjects and the institutional ethics committee of Government Medical College,

Kozhikode gave ethical approval on 11.06.2012.

INCLUSION CRITERIA

The first 30 newly diagnosed leprosy patients who successfully completed fixed duration

treatment from our tertiary care institution and who were willing to have a post-treatment skin

Study on the histopathology of leprosy skin lesions 143

biopsy were included in this study. Only those who underwent a pre-treatment biopsy from

our institution were included in the study.

EXCLUSION CRITERIA

Pure neuritic cases were excluded from the study.

A preset proforma was used to collect data regarding name, age, sex, clinical

manifestations including the number, morphology, distribution and size of skin lesions and

involved nerves with details of motor and sensory impairment from case records of individual

patients. Bacteriological and morphological indices in each case were carefully documented.

In our institution we routinely perform ear lobe smear and at least two slit-skin smears (from a

representative skin lesion and normal skin) for each leprosy patient and the Ziehl Neelsen

technique was used to stain the smear to determine the morphological and bacteriological

indices.5 The clinical spectrum determined on the basis of morphology and skin smear studies

(as per the Ridley-Jopling classification) was noted in each case. Clinical evidence of lepra

reaction when present was documented.6

Biopsies from the skin lesions were stained using haematoxylin and eosin to study the

morphology and Wade Fite technique to identify the acid fast bacilli (AFB).7

Histopathological features were documented in individual study subjects and the

histological classification was noted. Histological diagnosis of upgrading Type 1 lepra

reaction (T1R) was made when a biopsy revealed features of leprosy with the presence of a

richer infiltrate of protective cells such as lymphocytes and/or giant cells (with or without

dermal oedema) with respect to leprosy of a similar spectrum without reaction. Downgrading

T1R was diagnosed when histology revealed leprosy with a paucity of lymphocytes and/or

giant cells (with or without dermal oedema) with respect to leprosy of similar spectrum

without reaction. Dermal oedema alone without any increase or decrease in lymphocytes or

giant cells was considered as a T1R without upgrading or downgrading.2

Histology revealing neutrophil infiltration or neutrophil vasculitis on a background of

macrophage granuloma was taken as evidence of Type 2 lepra reaction (T2R).2

A final diagnosis was made on the basis of the clinical picture, skin smear studies and

histological features. Leprosy treatment as well as the treatment for lepra reactions received

by individual study subject were carefully documented.

At the time of completion of FDT, namely after 6 months (PB) or 12 months (MB), a

second incisional biopsy was taken from a site as near as possible to the pre-treatment biopsy

site. The two specimens (before and after FDT), were compared with respect to the basement

membrane pigmentation, percentage of dermis occupied by granuloma or inflammatory

infiltrate per average low power field, presence of protective cells such as lymphocytes and

epithelioid cells, the presence of acid fast bacilli and evidence of dermal fibrosis.

Histological diagnosis of T1R and T2R in post FDT specimens were made as described

for the pre-treatment biopsy.

Histological resolution achieved post treatment was classified as below:

Grade 1: Post-treatment specimen showing an increase in granuloma or inflammatory

infiltrate compared to pre-treatment specimen.

Grade 2: Post-treatment and pre-treatment specimens showing similar histology.

S. Sasidharanpillai et al.144

Grade 3: Post-treatment specimen showing persistence, but decrease in granulomas or

inflammatory infiltrate compared to pre-treatment specimen.

Grade 4: Post-treatment specimen showing resolution of all granulomas or complete

clearance of inflammatory infiltrate.

An attempt was made to study and compare the histological features before and after FDT

in PB, smear negative and smear positive MB cases.

Results

Among the study subjects 19 were males (Table 1).

The ages ranged from 11–70 years. Out of the 30 study subjects, the clinical diagnosis

was BT in 20, IL in two, LL in seven and BL in one. Clinical and histological concordance

was documented in 23 patients. Twenty six patients required MB treatment of which eight

were smear positive for AFB (Table 1).

Histological resolution achieved post treatment is as shown in Table 2.

Of the 22 patients with granuloma formation in the pre-treatment biopsy, two showed an

increase in granulomas after treatment. The first (case no 13, Table 1) had clinical and

histological evidence of TIR at the completion of treatment and was receiving systemic

steroids, while the post treatment biopsy of the second (case no 10, Table 1) revealed intra-

granuloma oedema and an increased number of lymphocytes, indicating T1R (Figure 1),

though there was no clinical evidence of lepra reaction.

In eight patients the post FDT skin biopsy in comparison to the pre-treatment specimen

showed a reduction, but persistence of granulomas. Two of them had clinical and histological

evidence of T1R at the completion of treatment (case nos. 23 & 29, Table 1) and one of them

was on prednisolone for the same (case no. 23, Table 1).

Another patient with persistence of granulomas (case no. 22, Table 1) after treatment had

ENL during FDT, which was managed by analgesics. She had no evidence of lepra reaction

clinically or histologically at the time of completion of treatment.

Three others had no clinical features to suggest lepra reaction at any time during FDT or

at the completion of FDT, but the histological analysis indicated upgrading TIR in the post-

treatment specimen of one patient (case no. 30, Table 1, Figure 2) and in both pre- and post-

treatment specimens of the other two (case no. 3 [Figure 3] & case no 17).

The remaining two of the eight patients who attained reduction, but not complete

clearance of granulomas post-treatment, had only a single granuloma (a marked reduction

from the pre-treatment status) at the time of completion of treatment (cases no. 8 & 19,

Table 1).

Five of the 12 patients whose post-treatment biopsy showed complete clearance of pre-

treatment granulomas leaving only a lymphohistiocytic inflammatory infiltrate (case no. 24,

Table 1) [Figure 4] had lepra reaction during FDT (four cases had T1R and one had T2R).

Two of them had clinical evidence of T1R (case no. 28, Table 1) and T2R (case no. 20,

Table 1) respectively at the time of post-treatment biopsy but there was no histological

evidence to suggest the same. Both had lepra reaction from the time of diagnosis and were

managed with prednisolone which was continued even after the completion of FDT due to

persistent reaction. The other three received prednisolone for T1R, during FDT which was

tapered off before release from treatment (RFT) and they had neither clinical nor histological

evidence of T1R or T2R at the completion of treatment.


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The post-treatment histological analysis revealed epithelioid granuloma formation in two

(case no 7 & 12, Table 1) of the eight patients whose pre-treatment histology showed only

inflammatory infiltrate (case no 7, Table 1) [Figure 5] whereas a marked reduction in

inflammation (post treatment) was noted in five (case no 6, Table 1) [Figure 6].

None of those without granulomas in the pre-treatment biopsy had clinical or histological

evidence of T1R during or at the completion of FDT, though six of them clinically belonged

to the BT spectrum.

6/8 without and 12/22 with pre-treatment granulomas had dermal fibrosis (thickened

fibrotic dermis with paucity of cells and adnexal structures) post-treatment (case no 25,

Table 1, Figure 7a, 7b).

Dermal fibrosis was more common in patients who achieved considerable post-treatment

reduction in granulomas or inflammatory infiltrate.

Another interesting observation was the post-treatment restoration of the reduced

basement membrane pigmentation noted in the pre-treatment biopsy of five patients

(Figure 8a, 8b).

Two were LL cases who attained complete clearance of pre-treatment granulomas. The

other three were BT, whose post treatment histology showed a reduction, but not complete

clearance of pre-treatment granulomas. One patient who showed loss of pigment in the

basement membrane zone over a granuloma before FDT (Figure 8c) regained the

pigmentation in the post treatment specimen (Figure 8d) with the clearance of the granuloma.

Figure 1. (a) Pre-treatment biopsy revealing epithelioid granuloma (H & E, X40). Inset: High power view of thegranuloma. (H & E, X400). (b) Post-treatment biopsy revealing increase in granulomas (H & E, X40). Inset: Highpower view of the granuloma showing increase in lymphocytes (H & E, X400). (c) High power view of the granulomain post-treatment biopsy revealing intra-granuloma oedema (H & E, X400).

Table 2. Histology resolution achieved after FDT in leprosy lesions

Histology resolution

Leprosy lesions withgranulomas in

pre-treatment biopsy

Leprosy lesions with nogranulomas in

pre-treatment biopsy Grand total

M F Total M F Total M F Total

Grade 1 1 1 2 2 0 2 3 1 4Grade 2 0 0 0 1 0 1 1 0 1Grade 3 6 2 8 2 1 3 8 3 11Grade 4 5 7 12 2 0 2 7 7 14Total 12 10 22 7 1 8 19 11 30


Figure 2. (a) Pre-treatment biopsy revealing macrophage granulomas (H & E, X40). (b) High power view of themacrophage granuloma showing foamy macrophages and few lymphocytes. (H & E, X400). (c) Wade Fite staining ofpre-treatment biopsy showing plenty of solid staining AFB (H & E, X1000). (d) Post-treatment biopsy revealingpersistence, but reduction of macrophage granulomas (H & E, X100). (e) High power view of the macrophagegranuloma showing foamy macrophages with plenty of lymphocytes and intra-granuloma oedema. (H & E, X400).(f) Wade Fite staining of post-treatment biopsy showing fragmented AFB (H & E, X1000).

Figure 3. (a) Pre-treatment biopsy revealing epithelioid granulomas (H & E, X100). Inset: High power view of thegranuloma showing intra-granuloma oedema, Langhan giant cell and many lymphocytes (H & E, X400). (b) Post-treatment biopsy revealing persistence, but reduction in granulomas (H & E, X40). Inset: High power view of thegranuloma showing increase in lymphocytes (H & E, X400).


Figure 4. (a) Pre-treatment biopsy revealing macrophage granulomas (H & E, X40). Inset: High power view of thegranuloma showing foamy macrophages (H & E, X400). (b) Post-treatment biopsy revealing complete clearance ofgranulomas leaving only minimal inflammatory infiltrate (H & E, X100).

Figure 5. (a) Pre-treatment biopsy revealing only inflammatory infiltrate (H & E, X100). (b) Post-treatment biopsyrevealing granulomas (H & E, X100). Inset: High power view of the granuloma (H & E, X400). (c) Another area ofpost-treatment biopsy revealing granulomas (H & E, X100). Inset: High power view of the granuloma (H & E, X400).


Figure 6. (a) Pre-treatment biopsy showing inflammatory infiltrate (H & E, X40). (b) Post-treatment biopsy showingmarked reduction in inflammatory infiltrate (H & E, X100).

Figure 7. (a) Pre-treatment biopsy revealing macrophage granulomas (H & E, X100). (b) Post-treatment biopsyrevealing complete clearance of granulomas leaving only minimal inflammatory infiltrate and dermal fibrosis(thickened fibrotic dermis with paucity of cells and adnexal structures) (H & E, X100).


Discussion

Our observation of an increase in granulomas in two patients with coexisting T1R at the

completion of treatment could be explained as due to the effective bacterial killing resulting

in improved immunity, enabling the host to mount a granulomatous response with abundance

of protective epithelioid cells and lymphocytes. Histological evidence of reaction in the

absence of the clinical features of the same, as observed in some of our patients, has been

reported earlier.6 Our observation of complete resolution of granulomas following FDT in

five of the seven patients who had lepra reactions (at the completion of FDT or during FDT)

treated with systemic steroids, could be attributed to the steroid induced immunosuppression

inhibiting the formation of new granulomas and promoting the clearance of the pre-existing

ones. A slow resolution of the granulomas that had formed as part of the reaction might have

contributed to the persistence of granulomas, in those who were not given systemic steroids.

Figure 8. (a) Pre-treatment biopsy revealing reduced basement membrane pigmentation (H & E, X400). (b) Post-treatment biopsy of the same patient showing restoration of basement membrane pigmentation (H & E, X400).(c) Pre-treatment biopsy revealing loss of pigmentation in an area of basement membrane above a granuloma(H & E, X400). (d) Post-treatment biopsy of the same patient showing reappearance of basement membranepigmentation (H & E, X400).


The appearance of granulomas after treatment in two patients who showed only

lymphocytic inflammatory infiltrate pre-treatment could be explained as due to the

bactericidal action of drugs enhancing the antigen exposure and inducing a granulomatous

reaction.

Our finding of the absence of clinical or histological evidence of lepra reaction in all

those whose pre-treatment biopsy showed only inflammatory infiltrate without granuloma,

warrants further study to assess whether the presence of granulomas in the pre-treatment

biopsy places a patient at greater risk for lepra reactions.

The two other prominent features noted in the post-treatment biopsy were dermal fibrosis

(18/30) and the restoration of the reduced basement membrane pigmentation noted in the pre-

treatment biopsy (5/30). Similar observations were made earlier.4 Others have noted dermal

fibrosis mainly in patients whose pre-treatment biopsies showed granulomas and not in those

who had only inflammatory infiltrate.4 Such an association was not found in our patients.

In our study, dermal fibrosis was associated with a near total clearance of pre-treatment

histological features. The exact reason for the hypopigmentation in leprosy lesions is not

clearly elucidated.8,9 Many have suggested defective transfer of melanosomes from

melanocytes to keratinocytes.8,9 The post-treatment reappearance of basement pigmentation

noted in five patients may be due to the correction of this transfer defect. Why this was limited

to five patients alone remains unknown. None of the patients without granulomas in the

pre-treatment biopsy, had reduction in basement membrane pigmentation. Whether the

inflammatory mediators released by granulomas play any role in inducing the reduction

or loss of pigment requires further analysis as the pre-treatment histology of one of our

patients showed loss of pigment of the portion of basement membrane overlying a granuloma

(Figure 8c) which reappeared after completion of treatment (Figure 8d) with resolution of the

granuloma.

The main limitation of our study was the small sample size, which makes it difficult to

interpret the data. Another drawback was that our study subjects included only patients who

were willing to have a post-treatment biopsy. So the 30 patients studied were not consecutive.

The study was conducted in a tertiary care institution with many referred cases and after

initiation of treatment some of them wanted to continue treatment from their nearest health

care institution; hence they were not included in the study. There was also a selection bias as

patients who suffered any complication during the treatment period, including reactions, were

more likely to agree to a post-treatment biopsy.

In spite of these limitations, we were able to study and analyse the histopathological

features of leprosy lesions after FDT. More prospective studies of the histology of leprosy

lesions before and after FDT may throw light on the several unknown aspects of this

disease.

References

1 Porichha D, Mukherjee A, Ramu G. Neural pathology in leprosy during treatment and surveillance. Lepr Rev,2004; 75: 233–241.

2 Sarita S, Muhammed K, Najeeba R et al. A study on histological features of lepra reactions in patients attending theDermatology Department of the Government Medical College, Calicut, Kerala, India. Lepr Rev, 2013; 84: 51–64.

3 Trindade MAB, Benard G, Ura S et al. Granulomatous Reactivation during the Course of a Leprosy Infection:Reaction or Relapse. PLoS Negl Trop Dis, 2010; 4: e921.

4 Joshy R. Clues to histopathological diagnosis of treated leprosy. Indian J Dermatol Venereol Leprol, 2011; 56:505–509.


5 Jopling WD, McDougall AC. The disease. In: Jopling WD, McDougall AC (eds). Handbook of Leprosy. 5th ed.,New Delhi: CBS Publishers 1996; pp. 11–53.

6 Lockwood DNJ, Nicholls P, Smith WCS et al. Comparing the clinical and histological diagnosis of leprosy andleprosy reactions in the INFIR cohort of Indian Patients with multibacillary leprosy. PLoS Negl Trop Dis, 2012; 6:e1702.

7 Azulay RD, Lygia MC, Andrade MD. Demonstration of Mycobacterium leprae in sections in 532 cases of leprosy.Comparative study between the Zeihl Klingmuller and the Wade Fite techniques. Int J Lepr, 1954; 195–199.

8 Westerhof W. A possible dysfunction of melanosome transfer in leprosy: an electron-microscopic study. ActaDerm Venereol, 1977; 57: 297–304.

9 Shereef PH, Thomas M. Hypopigmented macules in leprosy - a histopathological and histochemical study ofmelanocytes. Indian J Lepr, 1992; 64: 189–191.


histopathology of skin lesions of leprosy before and after ......macrophage granuloma was taken as...

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