“A COMPARATIVE STUDY OF ZIEHL-NEELSEN & MODIFIED

FITE-FARACO WITH AURAMINE RHODAMINE STAINING IN

DETECTION OF MYCOBACTERIUM LEPRAE IN TISSUE
SECTIONS”
by
Dr. DEEPA ADIGA. S. A

Dissertation submitted to the

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.

In partial fulfilment
of the requirements for the degree of
M. D.
in
PATHOLOGY

Under the guidance of

Dr. SUREKHA B. HIPPARGI MD
PROFESSOR, DEPARTMENT OF PATHOLOGY.

B.L.D.E.A’s
SHRI B. M. PATIL MEDICAL COLLEGE HOSPITAL & RESEARCH
CENTRE, BIJAPUR.

2010

i
 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation entitled “A COMPARATIVE STUDY

OF ZIEHL-NEELSEN & MODIFIED FITE-FARACO WITH AURAMINE

RHODAMINE STAINING IN DETECTION OF MYCOBACTERIUM

LEPRAE IN TISSUE SECTIONS” is a bonafide and genuine research work carried

out by me under the guidance of Dr. SUREKHA B. HIPPARGI MD, Professor,

Department of Pathology at B. L. D. E. A’s Shri. B. M. Patil Medical College

Hospital and Research Centre, Bijapur.

Date: Dr. Deepa Adiga. S. A

Place: Bijapur

ii
 CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “A COMPARATIVE STUDY

OF ZIEHL-NEELSEN & MODIFIED FITE-FARACO WITH AURAMINE

RHODAMINE STAINING IN DETECTION OF MYCOBACTERIUM

LEPRAE IN TISSUE SECTIONS” is a bonafide research work done by Dr. Deepa

Adiga. S. A, a post graduate student in Pathology and is done under my direct

supervision and guidance at B.L.D.E.A’s SHRI B.M. PATIL MEDICAL COLLEGE

HOSPITAL AND RESEARCH CENTRE, BIJAPUR in partial fulfilment of the

requirement for the degree of M.D. in PATHOLOGY.

Dr. SUREKHA B. HIPPARGI MD

Date: PROFESSOR,
DEPARTMENT OF PATHOLOGY,
B. L. D. E. A’s SHRI B. M. PATIL
Place : Bijapur MEDICAL COLLEGE HOSPITAL &
RESEARCH CENTRE, BIJAPUR.

iii
 ENDORSEMENT BY THE HOD AND PRINCIPAL

This is to certify that the dissertation entitled “A COMPARATIVE STUDY

OF ZIEHL-NEELSEN & MODIFIED FITE-FARACO WITH AURAMINE

RHODAMINE STAINING IN DETECTION OF MYCOBACTERIUM

LEPRAE IN TISSUE SECTIONS” is a bonafide research work done by

Dr.DEEPA ADIGA S A in partial fulfilment of the regulations of Rajiv Gandhi

University of Health Sciences, Bangalore for the award of the degree of M.D in

PATHOLOGY under the guidance of Dr. SUREKHA B. HIPPARGI MD,

Professor of Pathology at B. L. D. E. A’s Shri. B. M. Patil Medical College Hospital

and Research Centre, Bijapur.

Dr. B. R. YELIKAR M D Dr. M. S. BIRADAR MD

PROFESSOR & HEAD INCHARGE PRINCIPAL,
DEPARTMENT OF PATHOLOGY B. L. D. E. A’s SHRI B. M.PATIL
B. L. D. E. A’s SHRI B. M. PATIL MEDICAL COLLEGE HOSPITAL
MEDICAL COLLEGE HOSPITAL & & RESEARCH CENTRE,
RESEARCH CENTRE, BIJAPUR.
BIJAPUR.

Date: Date:
Place: Bijapur Place: Bijapur

iv
 COPYRIGHT

Declaration by the candidate

I hereby declare that the Rajiv Gandhi University of Health Sciences,

Karnataka shall have the rights to preserve, use and disseminate this dissertation/

thesis in print or electronic format for academic/ research purpose.

Date:
Dr. Deepa Adiga. S. A
Place: Bijapur

© Rajiv Gandhi University of Health Sciences, Karnataka

v
. . . . to my parents

vi
 ACKNOWLEDGEMENT

I take this opportunity to express my most humble and sincere gratitude to my

guide and teacher Dr. Surekha B Hippargi M.D. Professor of Pathology, Shri B.M.

Patil Medical College Hospital and Research Centre, Bijapur, for her invaluable

advice, constant guidance and motivation during this dissertation work and in pursuit

of my post graduate studies. I am obliged to her for her patience, co-operation,

encouragement and interest which she generously showed during the preparation of

this work.

I am very grateful to Dr. B.R. Yelikar M.D. Professor and Head, Department of

Pathology for his prudent and authoritative retrospective of this manuscript and

impassioned ebullience, which actuated me to furnish an adept study. I am deeply

indebted to him for his invaluable guidance.

My respectful thanks to Dr. B. V. Peerapur MD, Professor and Head,

Department of Microbiology for allowing me to use fluorescent microscope for the

study.

I am thankful to Dr. Surekha U. Arakeri M.D. Professor, Dr. R. M. Potekar

M.D. Professor, Dr. P.M. Patil M.D, Dr. Savitha Shettar MD, Dr. Ashwin MD, Dr. Mirza

Asif Baig MD, Dr. Anirudha MD, Dr. Mahesh Kumar MD, Dr. Mahendra MD, Dr.

Shilpa Patil MD, Assistant Professors and all other staff in the department of pathology

for their congenial supervision, assiduous concern and positive feedback, which has

made it conceivable for me to expedite this dissertation. I am also greatly thankful to

all the staff members of the department of surgery for their kind co-operation.

vii
 My deep gratitude to Dr. S.S. Jigjinni, Vice Chancellor and Dr. R.C. Bidri,

Principal, B.L.D.E.A’s Shri B.M.Patil Medical College Hospital and Research Centre,

Bijapur for permitting me to utilize resources in completion of this work.

I am extremely grateful to my senior postgraduates Dr Abdul and

Dr Madhavi for their valuable advice and help in completion of this study.

I remain grateful to my friends and well wishers Dr. Vijayalaxmi,

Dr Raghunath, Dr Guru, for their support and encouragement during this study.

I am very grateful to our technician’s G.I. Kabbin, Jagabatti, Jessy john and

Vidya who have helped me during this work.

I am deeply indebted to my parents, parents-in-law, my husband Dr

Gururaja Rao, my sister Divya, my son Sanat and all my family members for their

help, constant encouragement and moral support that led me to successfully complete

this dissertation work.

My special thanks to Dr. Mahesh H. Karigoudar M.D, Associate Professor,

department of pathology, B.L.D.E.A’s Shri B.M.Patil Medical College, Bijapur for

guiding and helping me in dissertation as well as building up and shaping up my

career.

My thanks to Om Sai Internet, Computers, Opp BLDE Hospital, Bijapur for

putting my dissertation work in a right format and in print. My sincere thanks to one

and all, who supported me for the completion of this dissertation.

Last but not the least, I convey my heartfelt gratitude to all the patients who

participated in my study without whose co-operation, this study would not have been

possible.

Date: Dr. DEEPA ADIGA S. A.

Place:Bijapur

viii
 LIST OF ABBREVIATIONS USED
(in alphabetical order)
AFB Acid fast bacilli
AR Auramine-rhodamine
BB Mid borderline leposy
BI Bacteriological index.
BL Borderline lepromaous leprosy.
BT Borderline tuberculoid leprosy.
DNA Deoxyribonucleic acid.
ENL Erythema nodosum leprosum.
FF Fite faraco stain.
FL Fluorescent stain
IL Indeterminate leprosy.
LL Lepromatous leprosy.
MB Multibacillary
PB Paucibacillary
PCR Polymerase chain reaction.
TT Tuberculoid leprosy.
WHO World Health Organisation
ZN Ziehl Neelsen

ix
 ABSTRACT

BACKGROUND: Leprosy is a chronic infectious disease caused by

mycobacterium leprae. In leprosy, histopathological examination of skin offers
the correct diagnosis. We evaluate fluorescent microscopy which is increasingly
used for rapid screening.

OBJECTIVES:

1. To compare the efficacy of auramine rhodamine stain (FS) with Ziehl-

Neelsen(ZN) and modified Fite-faraco(FF) staining in the diagnosis of
leprosy in tissue sections.
2. To assess the role of auramine rhodamine staining method in grading of
Hansen’s disease.

METHODOLOGY:Skin biopsies of sixty clinically diagnosed leprosy patients

received in the department of pathology were subjected to ZN stain, FF stain and FS.
Each case was evaluated for presence of bacilli as well as bacillary index (BI).

RESULTS: Sensitivity of FS for indeterminate, borderline tuberculoid leprosies were

100% each. Positivity rates and mean BI with FS was higher as compared to that of
ZN and Fite-faraco when the bacillary load was less (BI <3). There was significant
correlation between the three staining types at lower bacillary load. There was a
higher mean BI with fluorescent stain as well as detection of an additional
multibacillary case.

CONCLUSION: Fluorescent method is more sensitive than modified fite-faraco

method in detecting lepra bacilli in tissue sections especially in cases with BI<3. It
has an additional role in grading of Hansen’s disease based on BI. Hence it can prove
valuable in tissue sections with lower bacillary load where other methods fail.

KEY WORDS : Leprosy; Fluorescent microscopy; Modified Fite – Faraco stain; ZN

stain.

x
 TABLE OF CONTENTS

Sl. No. Particulars Page . No

1. Introduction 1

2. Objectives 3

3. Review of literature 4

4. Methodology 27

5. Observations 35

6. Discussion 50

7. Conclusion 56

8. Summary 57

9. Bibliography 58

10. Annexure

A. Proforma 64
B. Informed consent form 66
C. Master Chart 69

xi
 LIST OF TABLES

Sl. No Table Page No.

1 Age and sex distribution of patients. 35

2 Gender distribution of patients. 36

3 Different histological patterns in present study. 37

4 Percentage of histological diagnosis positive for ZN Stain. 38

5 Histological patterns and modified Fite-faraco stain. 39

6 Percentage of histological diagnosis positive for fluorescent 40

stain.
7 Comparison of positivity rates of ZN, Modified Fite-faraco and 41
fluorescent stains.

8 Histological findings and correlation of modified Fite-Faraco 42

stain with Ziehl-Neelsen and fluorescent stain.
9 Histological findings and correlation of mean bacillary index 43
among FF with ZN and FL.
10 Correlation between Fite faraco v/s Ziehl – Neelsen and 43
fluorescent method.
11 Bacillary index correlation among patients with lower BI (BI<3) 44
and higher BI (BI>3) between various staining types.
12 Cases showing upgrading of BI by fluorescent and ZN compared 45
to FF among PB and MB cases.
13 Comparison of shift in Ridley’s BI scale by FL stain and ZN stain 46
with that of FF among PB and MB cases.
14 Comparison of positivity rates of ZN staining, modified 51
Fite-faraco and fluorescent stain with that of other studies.
15 Comparison of positivity rates in various leprosy types by 53
modified FF and FL with that of other studies.

xii
 LIST OF FIGURES

Sl. No. Figure Page No.

1 Lepromatous leprosy, H&E 10X 47

2 Lepromatous leprosy, FF 100X 47

3 BL leprosy, H&E 10X 47

4 BL leprosy, ZN 100X 47

5 BT leprosy, H&E 10X 48

6 BT leprosy, H&E 10X 48

7 TT leprosy, H&E 10X 48

8 Indeterminate leprosy, AR 40X 49

9 BT leprosy, AR 40X 49

10 TT leprosy, AR 40X 49

11 Lepromatous leprosy, AR 40X 49

xiii
 INTRODUCTION

Leprosy is a chronic infectious disease caused by Mycobacterium leprae

which expresses itself in different clinicopathological forms, depending on the

immune status of the host.1

Mycobacterium leprae was discovered by Gerhard Henrik Armauer Hansen

in 1873.2 Leprosy causes permanent and progressive physical deformities in the

patient. It affects mainly the peripheral nerves. It also affects the skin, muscles,

eyes, bones, testes and internal organs. Leprosy is present in practically every corner

of the globe, but in tropical countries like India, it is still one of the problems of

public health importance.

This problem can be tackled by correct diagnosis and timely treatment. The

clinical diagnosis of leprosy has to be confirmed by diagnostic procedures like slit

skin smear and skin biopsy.

Histopathological examination of skin in leprosy exhibits different

morphological patterns depending on the immune status of the host. Ziehl-Neelsen

(ZN) staining method is the old and conventional method of detection of the organism

in clinical specimens3. Modified Fite-faraco technique is the routinely used method to

demonstrate Mycobacterium leprae in tissue sections.

Though modified Fite-faraco is more sensitive than Ziehl Neelsen method in

detection of Mycobacterium leprae in tissue sections, it is tedious, time consuming

and leads to observer fatigue.

Hence fluorescent microscopy has been used by some for rapid screening to

reduce observer fatigue and to increase sensitivity.

1
 This study was done to compare the sensitivity of fluorescent microscopy with

that of ZN staining and modified Fite-faraco technique in detecting Mycobacterium

leprae in tissue sections.

2
 AIMS AND OBJECTIVES

1. To compare the efficacy of auramine rhodamine with Ziehl- Neelsen and

modified Fite-Faraco staining in the diagnosis of leprosy in tissue sections.

2. To assess the role of auramine rhodamine staining method in grading of Hansen’s

disease.

3
 REVIEW OF LITERATURE

Historical Aspects:

Leprosy is the oldest disease known to mankind.4 G.H. Armauer discovered

Leprosy in 1873. He found that the cause of leprosy were the rods of bacilli in the

lesion. He had initially observed them in skin, nerves and visceral lesions

exhaustively in unstained tissue specimens and in due course he found that they could

be better visualized if treated with dilute osmic acid. 5

As the time progressed various methods were suggested and demonstrated by

different people to identify lepra bacilli in slit skin smears and in tissue sections.

Conventional ZN method of staining is one of the oldest methods of detecting

the organisms in tissue sections.3

The Fite methods are most commonly used for demonstrating lepra bacilli in

tissue sections. 6 Wade fite and Fite-faraco stains can demonstrate acid fast organisms

in tissue sections, which can also be demonstrated by fluorescence method and with

PCR technique. 7

In 1937 fluorescent microscopy for detecting Mycobacteria was first used by

Hagemann. 8

Following this in 1952, Gohar9 described the advantages of fluorescent

microscopy for detecting Mycobacterium leprae in smears.

In 1956, Khanolkar and Nerurkar10 used fluorescent microscopy in the

diagnosis of leprosy for smears.

4
 In 1960, Kuper and May11 introduced the fluorescent microscopy for the

detection of acid fast organisms in tissue sections. They added rhodamine to older

method of staining which improved contrast and better appreciation of bacilli by

fluorescent microscope.

In 1966, Silver12 et al, suggested various modifications in the procedure of

fluorescent staining. These included variation in duration of exposure to stain and

counterstain and mountant to be used.

Further in 1970, Mansifield8 observed that with the use of fluorescent

microscopy, the bacilli were easily and rapidly found. He also found that xylene

peanut oil mixture for deparaffinisation produces much brighter staining of the bacilli

in both fluorochrome and carbol-fuchsin stained tissue sections.

Jariwala and Kelkar 13 in 1979 observed that fluorescence method was superior

to the modified Fite-faraco method in detecting Mycobacterium leprae in tissue

sections particularly in paucibacillary cases. They also felt that the field covered was

16 times larger, so that an average section could, therefore, be scanned in two to three

minutes.

In 1987, Bhatia14 et al studied histopathological sections from cases of

indeterminate, tuberculoid and BT cases by fluorescent method. They found in their

study that fluorescent staining was superior to ZN staining and that it should

supplement ZN staining when decision regarding negativity of smear arises.

In 1988, Bhatia15 et al again conducted study on 84 skin smears and found that

75 smears showed AFB by auramine staining as compared to only 57 smears by ZN

staining. Also the BI by auramine staining was higher as compared to BI by ZN

5
staining. The study also revealed a minimal interobserver variation by auramine

method.

In 2002, Jain16 et al conducted a study on 715 clinical specimens, which

included sputum, fine needle aspirate, pus and body fluids and examined them by ZN

and AR staining techniques, simultaneously. They found sensitivity of auramine

rhodamine staining to be 98% as compared to 78.8% by ZN staining. Also fluorescent

stain was more advantageous in paucibacillary cases.

Nayak and Shivarudrappa17 in 2003, promoted fluorescent microscopy for

leprosy diagnosis after a study conducted in Victoria Hospital and Bowring and Lady

Curzon Hospital, Bangalore. They found a higher positivity rate with fluorescent

staining as compared to modified Fite-Faraco. The speed of observation and the

rapidity of finding the bacilli also reduce observer fatigue.

Conversely, Lacordaire18 in 1972 found the modified Fite-faraco method to be

superior compared with the fluorescent staining in detecting Mycobacterium leprae in

tissue sections.

Also, in 1981, Hardas 19and Lele, opined after their study on 117 smears and

69 biopsies that granular and dusty forms of the organisms were totally missed by

fluorescent method. Also high frequency of artifacts makes it less advantageous.

In the mean time many authors have studied different histological patterns in

tissue sections of skin biopsies of leprosy patients.

In a study conducted by Shenoi SD 20 et al in 1988, most common histological

pattern was borderline tuberculoid leprosy (50%) followed by tuberculoid

6
leprosy(22%), indeterminate leprosy(11%), borderline borderline(6%), lepromatous

leprosy(6%) and borderline lepromatous(5%).

Kar P.K21 et al, in their study, in 1994, found that the most common

histological pattern of leprosy was borderline tuberculoid leprosy(31.66%) followed

by indeterminate leprosy(29.16%), tuberculoid leprosy(18.33%), borderline

lepromatous leprosy(8.33%), borderline borderline leprosy(6.66%) and lepromatous

leprosy(5.83%).

EPIDEMIOLOGY

Approximately 2,96,499 people live in areas where leprosy is an important

problem. Leprosy continues to be a major public health problem in India with an

annual new case detection rate of 1.43 per 10,000 population. In India the prevalence

rate is 0.84 cases per 10,000 population. 22

Leprosy is known to occur at all ages from early infancy to very old age. Age

of onset is extremely variable with respect to time, place and country. 23

Although leprosy affects both the sexes, M: F ratio is 2:1. This could be

because leprosy workers are mostly men, the examination of women is less complete

and less satisfactory particularly in certain cultural situations resulting in under

detection of leprosy among females. It is also attributed to greater mobility and

increased opportunities for contact among men.22

Where leprosy treatment facilities exist, inactivation or cure is an important

mode of elimination from prevalence pool. Even in absence of specific treatment, a

majority of patients particularly of tuberculoid and indeterminate leprosy tend to get

cured spontaneously. A study in India has shown that over a period of 20 yrs, the

7
extent of spontaneous regression among children with tuberculoid leprosy was about

90%.

Bacterial Properties.

1. Taxonomical Classification

Class: Schizomycetes

Order: Actinomycetales

Family: Mycobacteriacae

Genus: Mycobacterium

Species: Leprae

2. Dimensions of the Bacilli

Length: 1-8 microns. They are slightly curved rod shaped bacilli with parallel

sides and rounded ends.

Width: 0.3 microns

Thickness of bacterial wall: 20 nm

3. General Properties 24, 25

It is acid fast, alcohol fast, gram positive, non-motile, non-spore forming,

obligate intracellular bacilli. They are commonly seen in the cytoplasm of the

macrophages and nerve bundles, either singly or in large bundles called as

globi.

8
4. Biological Properties:

They cannot be cultured in laboratory media. In mouse foot pad, doubling

time is 11-13 days. It divides by binary fission. Optimum temperature is

minus 27-30oC. Growth is decreased when tissue temperature is between 25o

to 36oC.

5. Biochemical Properties:

Capsule is composed of lipids which are partly responsible for electron

transparent zone. Cell wall consists of cross linked peptidoglycans covalently

attached to an arabino galactam polymer.

6. Laboratory methods of detection :

By microscopic examination of

a) Slit skin smear.

b) Nasal scraping.

c) Nose-blow smears.

d) Skin biopsies.

Stains used:

- Haematoxylin and eosin.

- Ziehl – Neelsen staining with 5% H2SO4.22

- Modified Fite-faraco stain. 26

- Auramine rhodamine fluorescent stain. 17

9
Bacteriological index 24, 27 (BI):

Shows the density of lepra bacilli in a smear or tissue section. BI includes

living, which are with solid staining and dead, which show fragmented or granular

bacilli. According to Ridleys logarithmic scale, it is graded from zero to six +, which

is based on the number of bacilli seen on an average microscopic field under 100 X

objective.

Average number of acid fast BI

Bacilli

0 bacilli in 100 fields O+

1-10 bacilli in 100 fields 1+

1-10 bacilli in 10 fields 2+

1-10 bacilli in a field 3+

10 -100 bacilli in a field 4+

100 -1000 bacilli in a field 5+

> 1000 bacilli in a field 6+

Paucibacillary BI 0-1 + Multi-bacillary BI >/= 2+

10
Auramine-Rhodamine staining technique and fluorescent microscopy. 17

Fluorescent microscopy has been used by some for rapid screening and to

reduce observer fatigue. There are few studies performed on tissue sections to detect

M. leprae by fluorescent microscopy. There are differing views about the sensitivity

of fluorescent microscopy in detecting M. leprae in tissue sections. In paucibacillary

cases this method has advantages over the modified Fite-faraco method and also that

it can be used as a supplementary tool when tissue sections stained by Fite-faraco

method fail to detect the bacilli.

8. Polymerase Chain Reaction:

The use of DNA amplification based on PCR provides or exquisitely sensitive

method for detecting M. leprae. This technique could be used to assess treatment of

paucibacillary patients and to detect presence or persistence of bacteria in detecting

sub-clinical infection. 25, 28

Studies using this technique have detected M. leprae DNA on swabs from

nasal mucosa of clinically normal individuals in a leprosy endemic population.

PATHOGENESIS AND IMMUNOLOGY OF LEPROSY29

Leprosy is characterized by well recognized pathological changes. These

pathological characteristics are strikingly different from other infectious diseases

because of unique features of M. leprae.

Commonly affected tissues are peripheral nerves and skin, rarely other tissues

like respiratory mucosa, lymph nodes etc. can be affected.

Leprosy patients, especially lepromatous patients are the main source of

infection. Bacilli are liberated into the environment through the oro-nasal sinuses and

11
skin ulcers of these patients. It is not absolutely certain how M. leprae enters the

human host.

Lepra bacilli first infect the neural tissue. Primary target are schwann cells.

Subsequently fate and type of lesion depend on immune states of the host. Bacilli

multiply within the schwann cell and perineural cells, later the bacilli destroy them.

Schwann cells liberate the bacilli, which enter the neighboring schwann cells and

spread the infection intraneurally. When the intraneural infection is recognized,

lymphocytes and macrophages infiltrate the nerve, later macrophages engulf the

bacilli. The bacilli multiply within the macrophages and then are carried to other parts

of the nerve and other nerves. Later they spread to other parts of the body through

blood, lymph and tissue fluids. 31 Experimental studies have shown two portals of

entry.30

a) Abraded skin at the cooler parts of the body.

b) Nasal mucosa.

Factors which influence the outcome of infection are age, skin, race, nutrition

and intercurrent disease. The major factor which determines the outcome is the

immune status of the host. 31

The macrophages become foamy due to utilization of oxygen and nutrition

from the cytoplasm, by the bacilli.

Later the macrophage ruptures, releasing the bacilli into the skin and other

structures. These bacilli are picked up by fresh macrophages. The body responds by

sending a number of lymphocytes and phagocytic macrophages to the site of

infection.

12
 In majority of the cases the bacilli are killed by the phagolysosome of the

macrophage and the infection fails to establish. In about 5% of cases the bacilli

multiply in the macrophage probably by preventing the formation of

phagolysosome.31, 32

Role of Immunology in Pathogenesis32:

There is involvement of cell mediated immunity and delayed hypersensitivity

in the pathogenesis of leprosy. These are responsible for the development of leprosy,

but it is the degree that determines the type of leprosy. This complete immune

response involves mainly T-lymphocytes, macrophages, to some extent B

Lymphocytes and the mediators.

The T-helper lymphocyte response to M. leprae determines whether an

individual has tuberculoid or lepromatous leprosy.

Patients with tuberculoid leprosy have a defective TH1 response or a dominant

TH2 response with production of IL-4, IL-5 and IL-10, which will suppress

macrophage activation.

In tuberculoid leprosy there are good number of CD4+T lymphocytes and in

lepromatous leprosy there are decreased CD4+T lymphocytes.

Tuberculoid leprosy - CD4+ T cells increase, CD8+ T cells decrease.

Lepromatous leprosy- CD4+ T cells decrease, CD8+ T cells increase.

In lepromatous patients, CD4+ T helper 2 cells (TH2 cells) when stimulated

by the antigen presenting cell secrete IL-4 and IL-5 which activate B-lymphocytes to

13
secreting plasma cells leading to formation of antigen – antibody complexes. This

causes type II reaction (Erythema Nodosum leprosum).

Classifications:

Classification of any disease, particularly leprosy, can be adjudged as

satisfactory only if it can be applied without much difficulty by different groups of

workers, that is clinicians, pathologists or immunologists. Between the different

groups, there should be a correlation of the criteria and the understanding must be

synchronized. 33

The ancient Indian medicine by Sushrutha Samhita in 600 BC described three

types – pure neuritic, non-lepromatous cutaneous lesions with sensory changes and

lepromatous cutaneous in which sensory changes are not prominent features. 34

Danielsen and Boeck (1848) divided leprosy into nodular and anesthetic types.

Hansen and Looft changed the anesthetic to maculoanesthetic types in 1895. Neisler

(1903) described three forms, namely - lepra tuberosa, lepra cutaneae and lepra-

nervosum. Jadussohn (1905) for the first time described the leprosy as tuberculoid.34

In 1931, Leonard Wood Memorial, Manila, Phillipines, divided leprosy into 3

types namely cutaneous, neural and mixed. The International Leprosy Congress,

Cairo (1938) adopted a classification in which the term ‘Cutaneous’ of the manila

classification was replaced by ‘Lepromatous’. 34

The second Pan American leprosy congress (1946) classified leprosy based on

histological grounds. Lepromatous was retained and neural was replaced by

tuberculoid, and the third type was named uncharacteristic. 34

14
 WHO (1952) classified leprosy into lepromatous, tuberculoid, borderline and

indeterminate. In 1955 Indian Association of leprologists classified leprosy into six

classes: lepromatous, tuberculoid, maculo- anesthetic, borderline, polyneuritic and

indeterminate. In revised Indian classification (1981) maculo--anesthetic type was

removed and included in tuberculoid type.35

In the same year Job & Chacko classified leprosy into lepromatous leprosy,

tuberculoid leprosy, borderline tuberculoid leprosy, borderline borderline leprosy,

borderline lepromatous leprosy, indeterminate leprosy and polyneuritic. 34, 35

Ridley and Jopling Immunological Classification (1966)

Ridley and Jopling (1966) suggested a classification system, which employed

correlation of clinical and histopathological status. 36

This classification is only for research purposes, according to Ridley and Jopling

themselves.37

1. Tuberculoid (TT)

2. Borderline Tuberculoid (BT)

3. Borderline Borderline (BB)

4. Borderline Lepromatous (BL)

5. Lepromatous Leprosy (LL)

15
WHO Clinical Classification 34, 38

This is a simple classification based on number of bacilli harbored in an individual.

1. Multibacillary [MB]

2. Paucibacillary [PB]

Bacteriological index if 2+ or more termed multibacillary and bacteriological

index less than that are termed paucibacillary.

This was modified in 1988 as multibacillary with bacterial index 1+ or more,

and paucibacillary with bacterial index 0+ [i.e., all smear negative]

Clinical Aspects of Leprosy Lesions

Leprosy, in some individuals, involves only one peripheral nerve

(mononeuritic) or causes a single skin blemish which persists indefinitely or

disappears on its own, while in others it produces multiple lesions or nodules, together

with polyneuritis and damage to vital organs, such as eyes, larynx, testes and bones.39

WHO, has set guidelines based on at least two of the following 41

1. Characteristic skin lesions.

2. Thickened nerves/AFB positive skin smear.

3. Sensory loss.

16
VARIOUS CLINICAL TYPES

1. Indeterminate Leprosy 31, 41, 42

Indeterminate leprosy is the earliest form of leprosy which manifest as small

one or few hypopigmented macules, about 1 cm or less than 5 cm in diameter, rarely

erythematous. Nerve thickening is usually absent. Even skin smears are usually

negative. The diagnosis is confirmed by histopathological examination. Indeterminate

leprosy may heal or remain indeterminate for a long period of time. It may sooner or

later progress to determinate forms of leprosy lesions.

2. Tuberculoid Leprosy 41, 42, 43

Commonly seen on face, dorsum of extremities and lower back, and affects

both skin and peripheral nerves. Lesions are usually single or two with well defined

borders, and are macular or rarely plaques. They may be hypopigmented or

erythematous with hair loss on their surface. Nerves are thickened with absence of

tenderness. Sensations of touch may be preserved. Skin smears are usually negative

for AFB.

3. Borderline Tuberculoid Leprosy 41, 42, 43

Commonest type of leprosy. Lesions may be single or multiple with varying

size and shape and are well defined, symmetrical with raised margins and hair loss.

Hypopigmentation and dryness are less severe than tuberculoid type. Satellite lesions

are seen near the edges of larger lesions.

Nerve thickening is present with asymmetry and there is loss of sensation over

the lesions.

17
 One of the striking features of borderline tuberculoid leprosy is tendency to

present with type I reaction. Most cases present as reaction. Skin smears sometimes

are positive for AFB.

4. Borderline Borderline Leprosy [Mid- Borderline] 41, 42

Most unstable and rare form. It spans the spectrum between lepromatous and

tuberculoid poles.

Multiple lesions of skin with varying size, shape and distribution are seen.

They may be macules, papules or plaques with ill defined margins, having moderate

hair loss. Nerve thickening is seen with asymmetry, with mild to moderate loss of

sensation. Skin smears are positive with many bacilli.

5. Borderline Lepromatous Leprosy 41, 42, 43

Classically the lesion start with macules, localized at first and later it is wide

spread as seen in lepromatous type. These macules are wide spread over the

extremities and lower back. Lesions are symmetrical and vary in size. Peripheral

nerve thickening is present with impairment of sensation. Skin smears are positive

with many bacilli often in clumps and globi.

6. Lepromatous Leprosy32, 41, 42, 43, 44

Lesions are seen all over the body with macules, papules or nodules and seen

over face, both upper and lower extremities and ears. They are symmetrical and are

slightly hypopigmented and sometimes erythematous with ill defined margins.

Sensations are slightly impaired with hairloss [leprous alopecia]. Nodular lesions

over the face coalesce together, with loss of eye lashes [madarosis] and depression of

nasal bridge [leonine facies]. Trophic ulcer formations may be seen in the extremities.

18
Muscle weakness and wasting may be seen. There may be involvement of eyes,

lymphadenopathy, testes or other systemic organs. Hands and feet may be swollen.

Involvement of mucosa of upper respiratory tract is seen in 80% of new lepromatous

cases.

Skin smears show plenty of AFB with multiple globi.

7. Pure Neuritic Leprosy 41

Also called as pure neural, primary neural, primary neurotic, primary

polyneuritic. Common in India. Presents with neurological deficit without any skin

lesions. It may present as anesthesia in an extremity or present with gradual foot drop.

Mono-neuritic is the most common form but multiple nerve involvement may be

present.

Histological Features of skin

Structure of skin.

Skin consists of 3 layers: Epidermis, Dermis and Subcutis.

Epidermis:

The epidermis derived from ectoderm, is a keratinizing stratified squamous

epithelium from which arises the cutaneous appendages, namely the pilosebaceous

follicles, nails, apocrine and eccrine sweat glands.

In addition to keratinocytes there is a ‘clear’ cell population, which includes

melanocytes and Langerhans’ cells.

19
The epidermis comprises five layers or strata:

• Keratin cell layer (stratum corneum)

• Clear cell layer (stratum lucidum)

• Granular cell layer (stratum granulosum)

• Prickle cell layer (stratum spinosum)

• Basal cell layer (stratum basale)

The basal cells are tall columnar cells aligned perpendicular to the basement

membrane and are the germinative cells of the epidermis and comprise stem cells and

proliferative cells.

Prickle cells are polygonal in outline, have abundant eosinophilic cytoplasm,

oval vesicular nuclei with conspicuous nucleoli.

Keratohyaline granules typify the granular cell layer. Further maturation leads

to loss of nuclei and flattening of keratinocytes to form the plates of the keratin layer.

Adjacent cells are united at their free borders by intercellular bridges (prickles or

desmosomes). It also unites the epidermis with the dermis is the basement membrane

region.

Melanocytes, of neural crest origin are usually located along the basal layer of

epidermis. The ratio of melanocytes to basal cells ranges from 1:4 on the cheek to

1:10 on limbs.

Langerhans cells are found within the supra basal layers of the epidermis and

also in the dermis. They represent potent stimulators of a wide range of T cell

mediated immune reactions.

20
Dermis:

The dermis or corium supports the epidermis and is composed of a fibrous

connective tissue component, collagen and elastic fibres in intimate association with

the ground substance.

Contained within the dermis are the epidermal appendages, blood vessels and

nerves and a cellular component including mast cells, fibroblasts, myofibroblasts and

macrophages. Smooth muscle is also represented in the erector pili muscles.

Sub cutis:

The sub cutaneous fat is divided into lobules by vascular fibrous septae and its

cells are characterized by the presence of a large single globule of lipid which

compress the cytoplasm and nucleus against the plasma membrane.45

Histological Features of Leprosy Lesions

Histological examination of all types of leprosy are done under following

criteria.41

1. Cell Type:

Lymphocytes are present in varying numbers in all leprosy lesions and

they are the predominant cell type in indeterminate leprosy.

Epithelioid cells and granulomas are found in tuberculoid types (BT and TT)

whereas foamy macrophages are predominantly seen in Lepromatous types (LL &

BL).

2. Bacterial Load:

Bacterial load is varied from almost absence of Mycobacterium leprae in

tuberculoid types to bacilli packed macrophages in lepromatous types.

21
3. Nerves:

Involvement of nerves and the presence of bacilli inside the nerves is also a

diagnostic feature.

I. Indeterminate Leprosy: 46, 47, 48

Majority of the times, clinical diagnosis of indeterminate leprosy is varied. To

make a definitive diagnosis, histopathological study is necessary.

Features are usually non-specific with epidermis showing no significant

change. But the dermis show mild lymphocytic and macrophage accumulation around

neurovascular bundles, superficial and deep dermal vessels, sweat glands and erector

pili muscle. Focal lymphocytic invasion into the lower epidermis and into the dermal

nerves may be observed. Schwann cell hyperplasia is a feature but it is highly

subjective. The diagnosis hinges on finding one or more acid fast bacilli in the sites

of predilection in nerve, in erector pili muscle, just under the epidermis or in a

macrophage about a vessel. Without demonstrating bacilli the diagnosis can only be

presumptive.

II. Tuberculoid Leprosy (TT): 43, 47, 48

Epidermis shows atrophy, occasionally few areas of hypertrophy seen.

Dermis is filled with granulomas containing aggregates of epithelioid cells even with

langhans type of giant cells. Granulomas almost replace the nerves, sweat glands, hair

follicles, erector pilorum muscles and sebaceous glands. These are surrounded by

dense lymphocytic infiltrate. There is no clear zone (Grenz zone) and the granulomas

are seen to hug the epidermis. Acid fast bacilli are rare and difficult to demonstrate.

22
III. Borderline Tuberculoid Leprosy (BT): 6, 46, 47

Atrophy of the epidermis is minimal depending on the size and extensiveness

of the granulomas. Dermis shows granulomas with peripheral lymphocytes that

follow the neurovascular bundles and infiltrate sweat glands and erector pili muscles.

Langhans giant cells are variable in number and are not large in size.

Granulomas along the superficial vascular plexus are frequent but they do not

infiltrate up into the epidermis. Nerve erosion and obliteration are typical.

Acid fast bacilli are scanty and are most readily found in the Schwann cells of

nerves.

IV. Borderline Borderline Leprosy (BB) Mid Borderline: 31, 46, 49

Rare type and is unstable and has atrophic epidermis. Dermis shows grenz

zone which is a clear zone which separates granulomas from the epidermis.

Granulomas are ill defined composed of mixture of good number of epithelioid cells,

scattered lymphocytes and few macrophages. Here the macrophages are uniformly

activated to epithelioid cells but are not focalized into distinct granulomas. There are

no langerhans giant cells.

Involvement of nerves also is seen with minimal destruction of the affected

nerves but reactive proliferation and edema of the perineurium is seen.

Acid fast Bacilli can be seen in schwann cells and in scattered macrophages.

V. Borderline Lepromatous Leprosy (BL) 31, 46, 49

Epidermis is always atrophic. Dermis shows mixture of many macrophages

with large number of lymphocytes, which are separated from the epidermis by a clear

zone (Grenz zone). Most of the macrophages are foamy with granular pink cytoplasm.

23
These inflammatory cells are also seen around hair follicles, sweat glands, sebaceous

glands and erctor pilorum muscles which may damage them.

There is also marked infiltration around the nerves, which show proliferation

of perineural cells with formation of onion skin perineurium (Concentric layers

around the nerves) on cross section.

Plenty of Acid fast bacilli can be demonstrated, which are distributed in

singles, clumps or occasionally in globi.

VI. Lepromatous Leprosy. 31, 46, 49

Has definite atrophy of epidermis with flattening of the rete ridges. Dermis

show band of cellular infiltration, consisting of majority of macrophages with few

lymphocytes. This layer is separated by a grenz zone from the epidermis.

Macrophages show vacuolated and foamy, pale cytoplasm. Few plasma cells may

also be seen. The macrophages also infiltrate around the hair follicles, sebaceous

glands and sweat glands. These structures appear atrophic.

Macrophages are also seen surrounding the nerves, but there is minimal

proliferation of the perineurium.

Large number of acid fast bacilli arranged in clumps (Globi) are seen in

macrophages, perineurium, schwann cells, sweat glands, sebaceous glands hair

follicles, erector pilorum muscle and even in endothelial cells.

VII. Histoid Leprosy.50

Histoid leprosy is an unusual form of lepromatous leprosy. The epidermis is

thinned by an expanding pseudoencapsulated dermal mass consisting of interlacing

bands and whorls of spindle shaped histiocytes. In early lesions, predominant cells

24
may be polygonal or irregular histiocytes. The immediate sub-epidermal zone

contains no infiltrate. The histoid masses contain unusually large numbers of acid fast

bacilli packed tightly in bundles and groups without disturbing cellular detail.

The lesion can resemble a dermatofibroma and must be differentiated from

other fibrohostiocytic and histiocytic skin tumors. It can be differentiated by

demonstrating intracellular acid fast organisms.

Histopathology of leprosy reactions6

1. Type-I Reaction:

Immunopathologic spectrum of leprosy is a continuum, patients may move

along it in both directions.

a) Upgrading Reaction: Shifts towards tuberculoid pole is called upgrading or

reversal reaction. The granuloma becomes more epithelioid and activated and

langhans giant cells are larger with increased lymphocytes. Important feature here is

edema within and about the granulomas.

Bacilli are decreased in number or absent. There may be fibrinoid necrosis

within granulomas & dermal nerves.

b) Down grading Reaction: Shifts toward the lepromatous pole is termed down

grading reaction. Even here, edema is the most important feature, but the granulomas

are disorganized with decrease in lymphocytic infiltration. There are good numbers

of macrophages with persistence of giant cells. Fibrinoid necrosis within granulomas

is less common. Over the time density of bacilli increases.

25
2. Type-II Reaction (Erythema Nodosum Leprosum- ENL)

Occur most commonly in lepromatous leprosy and less frequently in

borderline lepromatous leprosy. May be observed not only in patients under

treatment but also in untreated patients. Important feature here is dense infiltration of

polymorphs in the dermis and subcutaneous tissue with microabscess formation.

Damage to the elastic fibres and collagen is common. Rarely vasculitis or necrosis

and ulceration of skin are seen.

Bacilli are reduced where as foamy macrophages containing fragmented

bacilli are usual.

26
 MATERIALS AND METHODS

Source of data:

Skin biopsies from patients clinically diagnosed as leprosy were received in the

department of pathology, B.L.D.E.A’s Shri B.M.Patil Medical College Hospital and

Research Centre, Bijapur from August-2005 to July-2009.This included leprosy

patients attending the dermatology clinics of the hospital. Skin biopsies were obtained

after taking informed consent in all the cases.

Inclusion criteria

All cases clinically diagnosed as leprosy were included in the study.

Exclusion criteria:

1) Inadequate biopsy.

2) Bacillary fragments are not taken into consideration for diagnosis in case of

fluorescent microscopy.

Sample size : 60 cases

Method of collection of data:

Pertinent clinical history like age, duration of the lesion, site of the lesion,

significant family and personal history, history of associated diseases and any drug

intake were taken and entered in the proforma. After detailed general and local

examination, the site of the biopsy was selected. The selected patient’s consent was

taken after explaining the details of the biopsy procedure. The biopsy of the lesion is

done along with the surrounding area. The biopsy area is cleaned & painted with an

antiseptic solution and adequate amount of local anaesthetic (2% lidocaine) is injected

to the skin and subcutaneous tissue.

27
Biopsy Technique:

Punch biopsy was used for obtaining samples of skin biopsy. It is important to

select a proper site for biopsy. Biopsy was taken from the active lesion, after

injection of local anaesthetic. The specimen obtained with a 4mm biopsy punch was

used for histological study. A 3 mm punch was preferred for small lesions or biopsy

from face for cosmetic reasons. After that the skin specimen was loosened with the

biopsy punch instrument, dropped in a bottle containing 10% formalin, and sent to

histopathology laboratory.

The biopsy specimen provided included history of previous biopsies,

adequate clinical history and any special requests if required.

Gross examination of the skin biopsy:

The three dimensional size and shape of the skin biopsy was assessed

including the circular or elliptical shape of the biopsy.

The entire skin biopsy was submitted for routine processing and embedded in

paraffin wax. From each block, ribbons containing 4 serial sections each 5 microns

were taken. One section were taken for routine haematoxylin and eosin staining and

one each for ZN staining, fluorescent and Fite-faraco staining.

Details of the staining procedure.

5μm thick paraffin sections of the skin biopsy were stained with haematoxylin and

eosin.

HAEMATOXYLIN AND EOSIN STAIN:

a) Haematoxylin

b) Xylene I and II

c) Absolute alcohol I and II

28
 d) 90% alcohol

e) 1% eosin

Procedure:

1. Paraffin sections placed in xylene for 2 minutes.

2. Transferred to absolute alcohol for 1 minute.

3. Section drained and placed in 90% alcohol for 1 minute

4. Section transferred to haematoxylin for 10-40 minutes

5. Slides transferred to slide washing tray for blueing for 10 minutes

6. Section dipped in acid alcohol, agitated for few seconds for differentiation.

7. Section dipped in 1% eosin for 3 minutes and washed in water.

8. After draining, section transferred to 90% alcohol agitated for 10-15 seconds.

9. Slides transferred to absolute alcohol agitated for 10-15 seconds.

10. Slides transferred to absolute alcohol I and then to absolute alcohol II for 30

seconds.

11. Sections transferred to Xylene I and Xylene II until completely clear.

12. Sections mounted with DPX.

Results:

Nuclei – Blue. Cytoplasm – shades of pink

All the sections were examined under microscope. Pathological findings were noted at

the level of epidermis, dermis and sub-cutis and were segregated into different

histological patterns.

29
ZIEHL- NEELSEN STAIN.

a) Carbol fuchsin

b) 1% acid alcohol.

c) Methylene blue.

d) Xylene.

Procedure.

1. Paraffin sections placed in xylene for 30 min two changes each.

2. Sections were hydrated by passing through 90%, 70% and 50% alcohol.

3. Sections were stained with carbol fuchsin for 10 minutes.

4. Sections were decolorized in 1% alcohol.

5. Sections were washed in running water.

6. Sections were counterstained with methylene blue.

7. Slides were dried, cleared in xylene and mounted.

Results

Acid fast bacilli- Red , Background – Light blue.

All stained sections were screened with 40X objective. Sections showing

organisms with typical morphology of Mycobacterium leprae by the 40X objective

were confirmed using 100X objective. The typical rod shaped organisms which

stained red were taken positive. Bacteriological index was calculated under the oil

immersion field.

30
MODIFIED FITE FARACO STAIN

a) Carbol fuchsin

b) 1% acid alcohol.

c) Methylene blue.

d) 1 part Peanut oil & 3 part Xylene mixture.

e) Xylene

Procedure

1) Paraffin sections placed in Xylene & Peanut oil mixture 30 min two changes

each.

2) Drain off excess oil.

3) Blot the section lightly on filter paper 3 times.

4) Sections were stained with Carbol fuchsin for 20 minutes.

5) Sections were washed in running water for 5 minutes.

6) Sections were decolorized with 1 % acid alcohol.

7) Sections were washed in running water.

8) Sections were counterstained with methylene blue.

9) Sections were washed in running water for 5 minutes.

10) Sections were blotted and dried..

11) Sections were cleared in xylene and mounted.

Results

Acid fast bacilli – red, Background – light blue.

All stained sections were observed under 40X objective. Sections showing

organisms with typical morphology of Mycobacterium leprae by the 40X objective

31
were confirmed using 100X objective. The typical rod shaped organisms which

stained red were taken positive. Bacteriological index was calculated under the oil

immersion field.

FLUORESCENT STAIN

For fluorescent staining, sections were taken on clean scratch free glass slides

without egg albumin or any other adhesive. These tissue sections were stained with

fluorescent dye (Auramine-rhodamine) and examined under fluorescent microscope.

Procedure

Auramine rhodamine fluorescent stain as recommended by Kuper and May11

was used.

Following procedure was used.

1) Deparaffinization was performed with 1 part peanut oil and 3 parts xylene

mixture; two changes of 10 minutes each and then blotted carefully.

2) The slide was stained with filtered auramine rhodamine mixture at 650 C

minutes.

3) The slide was washed in running water for 2 minutes.

4) Decolorization was performed in 0.5 % hydrochloric acid in 70 % ethanol for

2 minutes.

5) The slide was washed under running water for 2 minutes.

6) Counterstaining was performed with 0.5 % aqueous potassium permanganate

for 2 minutes.

7) The slide was washed under running water for 2 minutes.

32
 8) Dehydration was performed in absolute alcohol by dipping the slide just once

and blot dried immediately.

9) The slide was mounted with glycerol using a scratch free cover slip.

Tissue sections were observed immediately under Carl Zeiss fluorescent

microscope, which had HBO 50 high pressure mercury short-arc discharge. Excitation

was with blue violet rays obtained with two BG 12 primary filters; an Abbe condenser

was also used. Each time the sections were screened, auramine-rhodamine stained

sections from a skin biopsy of a typical lepromatous leprosy patient and a skin biopsy

from a normal individual were used as controls.

All sections were screened with 10X and 40X objectives. Sections showing

organisms with typical morphology of Mycobacterium leprae bacilli by the 40X

objective were confirmed using 100X objective. Only solidly fluorescing organisms

were considered for a definitive diagnosis. Bacillary fragments were not taken into

consideration.

The typical morphology of the bacilli showing bright yellow fluorescence

emitted by the bacilli when interspersed with the artifact was considered the

diagnostic criteria for labeling the biopsy positive for Mycobacterium leprae.

Mycobacterium leprae appeared as rod shaped organisms that emitted bright yellow

fluorescence. Bacteriological index was calculated under oil immersion field.

33
Method of Statistical Analysis:

The following methods of statistical analysis have been used in this study. The

Excel and SPSS (SPSS Inc, Chicago, and Version 10.5) software packages were used

for data entry and analysis.

The results were averaged (mean + standard deviation) for each parameter for

continuous data and numbers and percentage for categorical data presented in Tables

and Figures.

Statistical analysis:

• Data represented by diagrammatic presentations and tabulations.

• Sensitivity, specificity, positive predictive value and negative

predictive values calculated.

• Data collected analysed using Chi-Square test. ‘p’ value of < 0.05 is

considered as statistically significant.

• Also data was analysed using Pearson Correlation (‘r’ value

determined) for comparison between groups.

34
 OBSERVATIONS

The present study was carried out on a total of 60 clinically diagnosed leprosy

patients attending the department of Dermatology, venereology and leprology of Shri

B M Patil Medical College , Bijapur from August 2007 to July 2009. The results

obtained after staining the biopsy slides with ZN stain, Modified Fite-faraco and

Fluorescent stain were analysed.

Table 1 : Age and sex distribution of patients.

Male Female Total

Age in Years
No % No % No %
< 20 5 15.6 5 17.9 10 16.7
21-30 5 15.6 11 39.3 16 26.7
31-40 5 15.6 6 21.4 11 18.3
41-50 7 21.9 5 17.9 12 20
>50 10 31.3 1 3.6 11 18.3
Total 32 100 28 100 60 100
Mean ± SD 42.6 ±16.76 30 ±13.52 36.7 ± 16.3

In the present study, patients in the age group of 21-30 years were affected

most with 16 cases (26.7%). The least affected age groups are those < 20 years,

comprising 10 cases (16.7%).

Age distribution of patients according to gender.

45
40 Male %
35
Female
30 %
Percentage

25
20
15
10
5
0
Up to 20 21-30 31-40 41-50 >50
Age in years

35
 Table 2: Gender distribution of patients.

Gender Number %
Male 32 53.3
Female 28 46.7
Total 60 100

In the present study males were affected the most, with 32 cases (53.3%) and females

being 28 cases (46.7%).

53% Female
Male 47%

36
 Table 3: Different histological patterns in present study.

HISTOPATHOLOGICAL Number
DIAGNOSIS (n=60) %
Indeterminate Leprosy 30 50
Tuberculoid Leprosy 2 3.3
Borderline Tuberculoid leprosy. 14 23.3
Borderline Borderline Leprosy 0 0
Borderline Lepromatous Leprosy 2 3.3
Lepromatous Leprosy 12 20
Total 60 100

In our study indeterminate leprosy was the most common constituting

30(50%) cases, followed by borderline tuberculoid leprosy 14(23.3%), lepromatous

leprosy 12(20%), borderline lepromatous 2(3.3%) and tuberculoid leprosy 2(3.3%).

There was no borderline borderline case in our study.

1.Indeterminate Leprosy
35
2.Tuberculoid Leprosy
30 3.Borderline Tuberculoid Leprosy
4.Borderline Borderline Leprosy
cases
No of

25
5.Borderline Lepromatous Leprosy
20 6.Lepromatous Leprosy
15

10

0
1 2 3 4 5 6
Histopathological diagnosis

37
 Table 4: Percentage of histological diagnosis positive for ZN Stain

Histopathological Total No. of No. of

Diagnosis patients Postives %
30
1.Indeterminate Leprosy 1 3.3
2
2.Tuberculoid Leprosy 0 0
14
3.Borderline Tuberculoid Leprosy 2 14.3
0
4.Borderline Borderline Leprosy 0 0
2
5.Borderline Lepromatous Leprosy 1 50
12
6.Lepromatous Leprosy 12 100
Total 60 16 26.7

In the present study various histological patterns showed varied positivity rates

for ZN stain. 1(3.3%) out of 30 patients of indeterminate leprosy, 2(14.3%) out of 14

cases of borderline tuberculoid leprosy, 1(50%) out of 2 cases of borderline

lepromatous leprosy and 12(100%) out of 12 cases of lepromatous leprosy were

positive by ZN stain. None of the tuberculoid leprosy cases showed positivity

with ZN stain.
1. IL
2.TT
3.BT
4.BB
14
5.BL
12 6.LL

10
No. of cases

0
1 2 3 4 5 6
Histopathological diagnosis & Ziehl-Neelsen stain

38
 Table 5: Histological patterns and modified Fite-faraco stain

Histopathological Total No. No. of

Diagnosis of patients Positives %
1.Indeterminate Leprosy 30 1 3.3
2.Tuberculoid Leprosy 2 0 0
3.Borderline Tuberculoid Leprosy 14 4 28.6
4.Borderline Borderline Leprosy 0 0 0
5.Borderline Lepromatous Leprosy 2 2 100
6.Lepromatous Leprosy 12 12 100
Total 60 19 31.7

In the present study various histological patterns showed varied positivity for

Modified fite faraco stain.

1(3.3%) out of 30 patients of indeterminate leprosy, 4(28.6%) out of 14 cases

of borderline tuberculoid leprosy,2(100%) out of 2 cases of borderline lepromatous

leprosy and 12(100%) out of 12 cases of lepromatous leprosy were positive by Fite-

faraco stain. None of the tuberculoid leprosy cases showed positivity with Fite-faraco

stain.

1.IL
14 2.TT
12 3.BT
No. of cases

10 4.BB
5.BL
8 6.LL
6
4
2
0
1 2 3 4 5 6
Histopathological diagnosis & modified Fite-Faraco Stain

39
Table 6: Percentage of histological diagnosis positive for fluorescent Stain

Histopathological Total No. of No. of

Diagnosis patients Postives %
1.Indeterminate Leprosy 30 8 26.7
2.Tuberculoid Leprosy 2 0 0
3.Borderline Tuberculoid Leprosy 14 4 28.6
4.Borderline Borderline Leprosy 0 0 0
5.Borderline Lepromatous Leprosy 2 2 100
6.Lepromatous Leprosy 12 12 100
Total 60 26 43.3
In the present study various histological patterns showed varied positivity for

fluorescent stain.

8(26.7%) out of 30 patients of indeterminate leprosy, 4(28.6%) out of 14 cases

of borderline tuberculoid leprosy, 2(100%) out of 2 cases of borderline lepromatous

leprosy and 12(100%) out of 12 cases of lepromatous leprosy were positive by

fluorescent stain. None of the tuberculoid leprosy cases showed positivity with

fluorescent stain.

1.Indeterminate Leprosy
2.Tuberculoid Leprosy
3.Borderline Tuberculoid Leprosy
4.Borderline Borderline Leprosy
14 5.Borderline Lepromatous Leprosy
6.Lepromatous Leprosy
12
10
No.of cases

8
6
4
2
0
1 2 3 4 5 6
Histopathological Diagnosis & Fluoroscent Stain

40
 Table7: Comparison of positivity rates of ZN, modified Fite-faraco and
fluorescent stains.

HISTOPATH Modified Fite-

OLOGICAL Total No. of Faraco Fluorescent
DIAGNOSIS patients ZN Stain method Method
Positivity rate Positivity rate Positivity rate
IL 30 3.3 3.3 26.7
TT 2 0 0 0
BT 14 14.3 28.6 28.6
BB 0 0 0 0
BL 2 50 100 100
LL 12 100 100 100
Total 60 26.7 31.7 43.3

Highest overall positivity rates were seen with FL (43.3%) compared to 31.7%

and 26.7% with FF and ZN methods respectively.

100 ZN Stain Positivity rate

Modified Fite-Faraco Positivity rate
90 Fluorescent method Positivity rate
80
1.IL
Percentage

70 2.TT
3.BT
60 4.BB
50 5.BL
6.LL
40
30
20
10
0
1 2 3 4 5 6
Histopathological diagnosis

41
 Table 8: Histological findings and correlation of modified Fite-faraco stain with
Ziehl-Neelsen and fluorescent stain

Histopathological Sensitivity Specificity PPV NPV

Diagnosis
ZN FL ZN FL ZN FL ZN FL
Stain Stain Stain Stain Stain Stain Stain Stain
1.IL 100 100 100 75.86 100 12.5 100 100
2.TL - - 100 100 - - 100 100
3.BT 50 100 100 100 100 100 83.33 100
4.BB - - - - - - - -
5.BL 50 100 - - 100 100 0 -
6.LL 100 100 - - 100 100 - -
Mean 75 100 100 91.95 100 78.125 70.8325 100

Considering Fite-faraco (FF) method to be the standard test, we compared the

performance of ZN and FL stain methods. FL stain showed 100% sensitivity as against

ZN which showed only 75% sensitivity compared to FF method. The apparent lower

specificity of FL method is due to its higher sensitivity as reflected in its higher positivity

rates compared to the FF stain (43.3% and 26.7% respectively), since we did not consider

any artifacts and non-solid bacilli as positive in our results.

42
 Table 9: Histological findings and correlation of mean bacillary index among
modified Fite-Faraco stain with Zeihl-Neelsen and fluorescent stain

Total No. Mean Bacillay Index

Histopathological
of FF ZN FL
Diagnosis
patients Stain Stain Stain
1.IL 30 1.1 0.8 1.2
2.TL 2 3 2.5 3.5
3.BT 14 1.2 1.2 1.4
4.BB 0 0 0 0
5.BL 2 3 4 4
6.LL 12 1.3 1.2 1.4
Total 60 9.6 9.7 11.5

Mean bacillary index of all the histological types with FF was 9.6, with ZN stain was

9.7 and with fluorescent stain was 11.5. This is in line with the higher sensitivity of

fluorescent method.

Table 10: Correlation between Fite faraco v/s Ziehl – Neelsen and fluorescent
method.

Fite-Faraco
Method
Pearson's 'r' P Value
Zeihl-Neelsen 0.96 <0.0001
Fluorescent Method 0.98 <0.0001

As we can see from Table 10. overall both Zeihl-Neelsen and fluorescent

methods show a statistically significant correlation with Fite-faraco method.

43
 Table 11: Bacillary index correlation among patients with lower BI (BI<3) and

higher BI (BI>3) between various staining types.

Method Fite- Faraco

BI<3 , Pearson’s ‘r’ BI>3 , Pearson’s ‘r’

Ziehl Neelsen -0.04 0.89
p=0.81 p<0.0001

Fluorescent method 0.73 0.84

(p<0.0001) (p=0.0004)

Since BI is a continuous variable we divided the cases into two groups ie.,

those with BI<3 and those with BI>3, for comparison between groups. Ziehl-Neelsen

method correlates well (r=0.89) with Fite- faraco method at higher BI (>3) but poorly

and insignificantly (p=0.81) so with lower BI (<3). However fluorescent method

retains good (r=0.73) and statistically significant correlation (P<0.0001) even at low

bacillary loads. Thus fluorescent method is more sensitive in detecting lepra bacilli in

cases with low bacillary load (BI <3).

44
Table 12: Cases showing upgrading of BI by fluorescent and ZN compared to FF
among paucibacillary and multibacillary cases.

BI PB cases MB cases Total

FL>FF 9 2 11

FL<FF 0 1 1

Net additional 9 1 10
cases detected by
FL

BI PB cases MB cases Total

ZN>FF 1 0 1

ZN<FF 1 7 8

Net additional 0 -7 -7
cases detected by
FL

Among paucibacillary cases FL stain shows a higher BI compared to FF in 9

cases, while among multibacillary cases, only 1 additional case had a higher BI

compared to FF.

No net additional case could be detected by ZN stain compared to FF. In fact

ZN stain showed a lesser BI compared to FF among 7 multibacillary cases proving

inferiority of ZN stain compared to FF stain.

45
Table 13 : Comparison of shift in Ridley’s BI scale by FL stain and ZN stain with
that of FF among paucibacillary and multibacillary cases.

BI FF pauci FF multi Net FF pauci FF multi Net

upgradation upgradation
to to of Ridley’s to to of Ridley’s
scale. scale.
FLmulti FLpauci ZNmulti ZNpauci

No. of 1 0 1 1 2 -1
cases

Use of FL stain resulted in diagnosis of an additional case of multibacillary

while ZN stain failed to correctly classify one case of MB as diagnosed by FF stain.

This ability of FL stain to diagnose additional cases of multibacillary cases has

implications in therapy and follow-up.

46
47
48
49
 DISCUSSION

Leprosy continues to be a major public health problem in India with a annual

new case detection rate of 0.84 per 10, 000 population.22 Leprosy affects skin,

peripheral nerves and other organs directly or indirectly, leading to progressive and

permanent deformities in the patients. Clinical presentations are varied with so many

diversities between the clinical and histopathological features.

Histopathological examination is the keystone in the diagnosis and

categorization of leprosy. Modified Fite-faraco technique is the routinely used method

to demonstrate mycobacterium leprae in tissue sections. Detection of Mycobacterium

leprae in tissue sections by modified Fite-faraco is tedious, time consuming and leads

to observer fatigue. Hence fluorescent microscopy has been used by some for rapid

screening, to reduce observer fatigue and to increase sensitivity.

There is an increasing need for evaluation of newer techniques for the detection

of mycobacterium leprae to achieve rapid screening and reduce observer fatigue,

while increasing sensitivity and specificity.

In the present study we compare the performance of fluorescent microscopy,

modified fite faraco and ZN stains in detecting Mycobacterium leprae in tissue

sections.

Skin biopsies of 60 patients (32 males and 28 females) clinically diagnosed as

leprosy was studied.

Most patients (26.7%) were between 21 to 30 years. Indeterminate leprosy was

the most common histological type (50%) followed by borderline tuberculoid

leprosy(23.3%).

50
 Table 14: Comparison of positivity rates of ZN staining, modified Fite-faraco
and fluorescent stain with that of other studies.

Various studies ZN stain Fite-Faraco Fluorescence

procedure method
No. of Positive No. of Positive cases No. of Positive

cases cases

Present study 16(26.7%) 19 ( 31.7 % ) 26 ( 43.3 % )

Mukkamil AS et - 25 ( 44.64 % ) 39 ( 69.64 % )

al17

Jariwala et al13 - 20 ( 40.0 % ) 22 ( 44.0 % )

Bhatia et al15 57(67.8%) - 75(89.2%)

Lacordaire Lopes - 26 ( 86.6 % ) 10 ( 33.3 % )

de Faria18

The present study shows a higher positivity rate in detecting the bacilli with

fluorescent staining as compared to that of modified Fite-faraco which is comparable

to the studies done by Mukkamil AS et al17 and Jariwala et al.13

Also, in the present study, the positivity rate with ZN staining is lower as

compared with that of fluorescent staining. A study done by Bhatia et al15 which

showed more positives cases by fluorescent method as compared to that by ZN stain

though they did not use modified Fite-faraco in their study.

Thus our study shows that fluorescent stain is better than both modified Fite-

faraco and ZN stain in detecting lepra bacilli in tissue sections.

51
 In a study by Lacordaire Lopes de Faria18 positivity rates with modified Fite-faraco

is higher than that with fluorescent microscopy. In his study he used egg albumin as

adhesive and phenol which produces considerable artifacts. He found the presence of

artifacts from albumin and phenol to be a major problem. We did not face such problems

because neither egg albumin nor any other adhesive was used. The bacilli however could

be easily differentiated because the non specific artifact has pale yellow fluorescence,

where as the bacilli have bright yellow fluorescence.

52
 Table 15: Comparison of Positivity rates in various leprosy types by modified

Fite- faraco and flourescent methods with that of other studies.

Mukkamil A.S Jariwala H. J

Leprosy et al17 et al13 Present Study
types FF vs FL FF vs FL FF vs FL
Positivity rate Positivity rate Positivity rate
Difference Difference Difference
IT 32% 0% 23.4%
TT 33% 0% 0%
BT 17% 9% 0%
BB * ** 0%
BL * 0% 0%
LL 0% 0% 0%
82% 13% 11.6%

17
* There were no BB and BL cases in the study by Mukkamil et al.

**There were no BB cases in our study.

In the study done by Mukkamil17 et al. positivity rate difference between FF

and FL stains were higher in TT, whereas in our study the difference was higher in IT.

This could be because of insufficient cases of TT in our study, leading to insufficient

sample size for statistical evaluation.

However from the present as well as other studies it is evident that the

positivity rate with fluorescent stain was more as compared to modified Fite Faraco.

Furthermore the higher positivity rates with FL stain were seen in cases with lower

bacillary load while the difference evened out with LL and BL cases where the

difference in the positivity rates are nil. This highlights the superiority of fluorescent

stain especially in cases with lower bacillary load.

53
 Bacillary index correlation of Ziehl-Neelsen and fluorescent method with Fite –
faraco.

Overall both Ziehl Neelsen method and fluorescent method correlate

significantly (P<0.0001 in both and r= 0.96 and 0.98 respectively). However when we

look at groups with lower (<3) and higher BI (>3), fluorescent method retains good

(r=0.73) and statistically significant correlation (P<0.0001) even at low bacillary

loads; However Ziehl Neelsen method shows poor (r=-0.03) and insignificant

correlation with Fite- Faraco method (p=0.81) with lower BI (<3). This is similar to

the observation seen with different histopathological types, where fluorescent method

retains useful sensitivity even in histopathological types with lower bacillary load.

Hence fluorescent stain has an added advantage of its usefulness in assessing

bacterial index needed to categorise leprosy especially at lower bacillary load, apart

from its higher case pickup rate. This can have implications in catergorising a case as

paucibacillary v/s multibacillary, having treatment implications.

Bacillary Index by Zeihl-Neelsen and Fluorescent Method

BI_FL
3
BI_ZN

-1
0 1 2 3 4 5 6
Bacillary Index by Modified Fite-Faraco Method

54
 Also among paucibacillary cases, in particular, FL stain shows a higher BI

compared to FF stain. Hence FL stain is more useful in detecting Hansen’s bacilli

among paucibacillary cases.

Added to this, with FL, there is a shift of paucibacillary to additional

multibacillary cases that has implications in therapy and followup.

Hence fluorescent stain has an added advantage of its usefulness in assessing

bacterial index needed to categorise leprosy especially at lower bacillary load, apart

from its higher case pickup rate.

55
 CONCLUSION

• Fluorescent microscopy has higher case pick-up rates when compared to Ziehl

–Neelsen and modified Fite-faraco stains as evident by its higher sensitivity.

• Fluorescent microscopy is more reliable for bacterial indexing as compared to

modified Fite-faraco and ZN stain especially in low bacillary load (BI

<3+)which is very important for precise categorization of leprosy and hence

treatment.

• Fluorescent microscopy can be used as a supplementary tool when tissue

sections stained by modified Fite- faraco method fail to detect the bacilli in

tissue sections or categorise as paucibacillary cases.

• The procedure is valuable in cases where negativity of sections is to be

certified.

56
 SUMMARY

Skin biopsies from 60 leprosy patients were received in the department of

pathology, B.L.D.E.A University Shri B. M. Patil Medical College Hospital, Bijapur

from August-2005 to July-2009.

Each case was evaluated for the presence of acid fast bacilli and bacterial

index, after staining with H&E, Ziehl-Neelsen , modified Fite-faraco and auramine-

rhodamine stains.

Maximum number of patients were in 3rd decade, least affected being those

< 20 years. Males were affected more compared to females. Indeterminate leprosy

was the most common histological type, and borderline borderline least common.

Positivity rate with fluorescent stain was 43.3%, whereas with ZN and FF

were 26.7% and 31.7% respectively. Also the mean bacillary index with FF was 11.5

which was higher than that of ZN and FF. Both FL and ZN correlated significantly

(p<0.005) with the standard FF. However, FL did so at BI<3 which ZN failed to. FL

stain showed a higher bacillary index in a net of 10 cases as compared to FF, whereas

ZN showed a lower BI in 7 cases. Staining by fluorescent method detected an

additional multibacillary case which was categorized as paucibacillary by FF.

Hence apart from its higher probability of detecting a case, fluorescent

microscopy has an additional value in more accurate grading of Hansen’s disease,

which affects therapy and outcome.

57
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63
 PROFORMA

NAME : IP No. :

AGE : Date :

ADDRESS : Ref. by :

Chief complaints :

History of present illness :

Past history :

Treatment history :

Physical examination :

General physical examination:

Systemic examination :

Local examination :

Number :

Size :

Colour :

Sensation :

Nerves :

Investigations :

Hb%

ESR

64
TC

DC

Clinical diagnosis :

Histopathological diagnosis by :

1. Haematoxylin and eosin :

2. Zeihl-Neelsen stain :

3.Modified Fite-Faraco stain :

4. Auramine rhodamine stain :

65
 B.L.D.E.A’s SHRI. B.M. PATIL MEDICAL COLLEGE HOSPITAL AND

RESEARCH CENTER, BIJAPUR-586103

RESEARCH INFORMED CONSENT FORM

TITLE OF THE PROJECT : A COMPARATIVE STUDY OF ZIEHL-

NEELSEN & MODIFIED FITE-FARACO

WITH AURAMINE RHODAMINE

STAINING IN DETECTION OF

MYCOBACTERIUM LEPRAE IN TISSUE

SECTIONS.

PRINCIPAL INVESTIGATOR : DR. DEEPA ADIGA.S.A.

P.G.DEPARTMENT OF PATHOLOGY

P.G. GUIDE : DR.SUREKHA.B.HIPPARGI

PURPOSE OF RESEARCH :

I have been informed that this study is done to know the diagnostic utility of

auramine rhodamine stain in clinically diagnosed cases of leprosy.

PROCEDURE:

I understand that, I will undergo detailed history and clinical examination after

which skin biopsy will be taken and will be subjected to pathological study.

RISK AND DISCOMFORTS:

I understand that, there is no risk involved in the procedure performed.

BENEFITS:

I understand that my participation in the study will help to know the diagnosis

of the lesion within a short time after skin biopsy.

66
CONFIDENTIALITY:

I understand that the medical information produced by the study will become a

part of hospital record and will be subjected to confidentiality and privacy regulations

of the hospital. If the data is used for publications the identity of patient will not be

revealed.

REQUST FOR MORE INFORMATION:

I understand that my participation is voluntary and I may refuse to participate

or withdraw from the study at any time.

INJURY STATEMENT:

I understand that in the unlikely event of injury to me during the study I will

get medical treatment but no further compensations.

I have read and fully understood this consent form. Therefore I agree to

participate in the present study.

_____________________ _______________

Participant / Guardian Date:

_____________________ _______________

Signature of Witness Date:

I have explained the patient the purpose of the study, the procedure required

and possible risk and benefit to the best of my ability in the vernacular language.

____________________ _______________

Investigator / P.G. Date:

____________________ _______________

Witness to Signature Date:

67
STUDY SUBJECT CONSENT STATEMENT:

I confirm that Dr. Deepa Adiga S A, has explained to me the purpose of

research, the study procedure, that I will undergo and the possible discomforts as well

as benefits that I may experience in my own language. I have been explained in my

own language and I understand the same. Therefore I agree to give consent to

participate as subject in this research project.

(Participant) Date

(Witness to signature) Date

68