1

DIAGNOSIS OF KALA-AZAR FROM BUFFY COAT USING DIFFERENT TECHNIQUES INCLUDING

POLYMERASE CHAIN REACTION (PCR)

DR. RISHDINA MIRZA M.B.B.S

DEPARTMENT OF MICROBIOLOGY MYMENSINGH MEDICAL COLLEGE

MYMENSINGH, BANGLADESH

JULY 2010

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TO WHOM IT MAY CONCERN

This is to certify that Dr. Rishdina Mirza, a student of thesis part has completed

the thesis entitled �Diagnosis of Kala-azar from buffy coat using different

techniques including Polymerase chain reaction (PCR)� in the Department of

Microbiology, Mymensingh Medical College under my guidance and supervision

and this is up to my satisfaction. Her protocol was approved by protocol approval

committee of the Department of Microbiology and Ethical review committee of

Mymensingh Medical College.

Dated, Mymensingh The

Professor Dr. Md. Akram Hossain Head of the Department of Microbiology

Mymensingh Medical College Mymensingh, Bangladesh

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ACKNOWLEDGEMENTS

First of all thanks to Almighty Allah, the merciful and the beneficent, for giving

me the opportunity and providing me the ample energy and patience to complete

the entire thesis work.

I am very grateful and deeply indebted to my honourable teacher and guide

Professor Dr. Md. Akram Hossain, Head of the Department of Microbiology,

Mymensingh Medical College. It is my great pleasure to express my deepest

regards and whole hearted indebtedness to him for his inspiring encouragement,

continuous guidance, active cooperation, constant supervision, valuable

suggestions, constructive criticism and help in carrying out this work successfully.

I convey my deepest regards and gratitude to my respected teacher Professor Dr.

A.K.M. Shamsuzzaman, Head of the Department of Microbiology, Begum

Khaleda Zia Medical College, Dhaka for his cordial cooperation and valuable

suggestions.

I would like to express my gratitude to Dr. Md. Chand Mahmud, Assistant

Professor, of the Department of Microbiology, Mymensingh Medical College for

his valuable suggestions. My cordial respect and complements to Dr. Shyamal

Kumar Paul, lecturer of the Department of Microbiology, for his cooperation and

efforts for this thesis. I am thankful to the honorable members of the Ethical

review committee for giving kind approval of my thesis protocol. I am also

obliged to Professor Dr. Md. Aminul Haque, Principal, Mymensingh Medical

College, for his kind permission to conduct the present study in this institution.

I express my grateful thanks to Dr. Md. Abdur Razzaque Mia, Associate Professor

and Head of the Department of Biochemistry, Mymensingh Medical College, for

his cordial cooperation and valuable suggestion.

I would like to express my gratitude to all other Lecturers, M.Phil Students in the

Department of Microbiology, Mymensingh Medical College.

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I would like to thanks Abu Hanif Md. Al-Rafi, Laboratory Technologist and also

thanks to all other Laboratory Technologists and supporting staffs of

Microbiology Department, Mymensingh Medical College. I must thanks to

Librarian of Mymensingh Medical College for providing me journals, books and

necessary articles.

I owe my heartfelt gratitude and sincere thanks to my brother Mirza Masrur Huq

my sister Dr. Nadia Mirza and my brother-in-low Dr. Sirazul Kader, for their

affectionate support.

I would like to convey my greatest regards and sincere thanks to my dear husband

Dr. Md. Mazharul Alam for his patience, devoted cooperation and assistance in

collecting specimens and sensible sharing of my pains and pleasures.

I remained incomplete if I do not express wholehearted thanks and gratitude to my

beloved mother Dr. Anwara Khanam, for her blessings and sparing me so much

time in this job from all sorts of social and familial responsibilities.

Last but not the least, I am indebted to those cases and controls form whom I have

collected specimens and pray to Almighty for their quick recovery.

Lastly my profound gratitude and heartfelt regards to my beloved father Prof. Dr.

Mirza Hamidul Huq, ex. Principal, Mymensingh Medical College, Mymensingh

of who always inspired me for higher studies and is my constant source of

inspiration and encouragement in every step of my life.

Thanks again to all.

Mymensingh, July 2010 Dr. Rishdina Mirza

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CONTENTS

PAGE NO.

LIST OF TABLE vii

LIST OF FIGURE viii

LIST OF ABBREVIATION IX

SUMMARY xiii

CHAPTER 1 INTRODUCTION 1

OBJECTIVES 7

CHAPTER 2 REVIEW OF LITERATURE 8

CHAPTER 3 MATERIALS AND METHODS 52

CHPATER 4 RESUTLS 63

CHAPTER 5 DISCUSSON 85

CHAPTER 6 CONCLUSION 93

BIBLIOGRAPHY 94

APPENDICES 127

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6

LIST OF TABLES

TABLE NO. TITLE PAGE NO.

Table 1 Age and sex distribution of subjects 64

Table 2 Housing condition among study population 66

Table 3 Comparison between low and middle socioeconomic status in Kala-azar patients 68

Table 4 Duration of fever in Kala-azar cases 70

Table 5 Hepato-splenomegly in Kala-azar cases 72

Table 6 Haematological findings of (41) confirmed cases of kala-azar 74

Table 7 Prevalence of kala-azar cases among study population 76

Table 8 Sensitivity and specificity of LD body microscopy from buffy coat in confirmed KA cases 78

Table 9 Sensitivity and specificity of buffy coat PCR in confirmed KA cases 80

Table 10 Sensitivity and specificity of buffy coat microscopy in ICT positive KA cases 82

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LIST OF FIGURES

FIGURE NO. TITLE PAGE NO.

Figure 1 Distribution of kala-azar cases in hospitals 84

Figure 2 Buffy-coat 139

Figure 3 Buffy-coat 139

Figure 4 Buffy-coat smear 140

Figure 5 Leishmania donovani in buffy coat smear at 100X (oil immersion) lens under microscope 141

Figure 6 Leishmania donovani in buffy coat smear at 100X (oil immersion) lens under microscope 141

Figure 7 Leishmania donovani in buffy coat smear at 100X (oil immersion) lens under microscope 142

Figure 8 Leishmania donovani in buffy coat smear at 100X (oil immersion) lens under microscope 142

Figure 9 PCR amplification product of Leishmania donovani from buffy coat samples, Lane MW = 100 bp DNA ladder, Lane 1-15 patients buffy coat sample. Products were analyzed by electrophoresis in 1% agarose gel containing 0.5 g of ethidium bromide/ml in TBE buffer and photographed under UV illumination. 143

Figure 10 PCR amplification product of Leishmania donovani from buffy coat samples, Lane MW = 100 bp DNA ladder, Lane 1-8 patients buffy coat sample; Lane NC = Negative control. Products were analyzed by electrophoresis in 1% agarose gel containing 0.5 g of ethidium bromide/ml in TBE buffer and photographed under UV illumination. 144

Figure 11 rK39 immunochromatographic dipstick test for kala-azar 145

Figure 12 Bone marrow smears showing amastigote form of Leishmania donovani 146

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LIST OF ABBREVIATIONS Abbreviation Expanded version

ACP = Acid phosphatase

AIDS = Acquired immune deficiency syndrome

AMP = Adenosine monophosphate

APC = Antigen presenting cell

AQP = Aquaglyceroprotein

AT = Aldehyde test

ATP = Adenosine triphosphate

bp = Base pair

CCIEP = Counter-current immunoelectrophoresis

CD = Cluster of differentiation

CFT = Complement fixation test

CL = Cutaneous Leishmaniasis

cm = Centimeter

CRP = C-reactive protein

CSF = Colony stimulating factor/Cerebrospinal fluid

DAT = Direct agglutination 0C = Degree centigrade

DDT = Dichloro Diphenyl Trichloroethane

DGHS = Director General for Health Services

DNA = Deoxy ribonucleic acid

dNTP = Deoxynucleotide triphosphate

DTH = Delayed type of hypersensitivity

EDTA = Ethylenediamine tetra-acetic acid

ELISA = Enzyme linked immunosorbant assay

et al = at alia (all others)

FCS = Foetal calf serum

GBP = Gene B protein

GCS = Gamma-glutamylcysteine synthetase

G-CSF = Granylocyte colony stimulating factor

GKO = Gene knockout

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LIST OF ABBREVIATIONS (CONTD.) Abbreviation Expanded version

GM-CSF = Granulocyte-Macrophage colony stimulating factor

gp = Glycoprotein

Hsp = Heat shock protein

HCl = Hydrochloric acid

ICDDRB = International Center for Diarrhoeal Diseases and Research, Bangladesh

ICT = Immunochromatography test

IFAT = Indirect fluorescent antibody test

IgG = Immunoglobulin G

IL = Interleukin

INF = Interferon

IU = International unit

KA = Kala-azar

KCl = Potassium chloride

kDa = Kilo-dalton

kDNA = Kinetoplast DNA

Kg = Kilogram

L = Leishmania

LAAMB = Lipid associated amphotericin B

LACK = Leishmania homologue of receptor for Activared C Kinase

LAT = Latex agglutination test

< = Less then

LD bodies = Leishmania donovani bodies

LdMT = L. donovani miltefosine transporter

LdRos = L. donovani Ros protein

LMDR = Leishmania multi-drug resistant

Ln-PCR = Leishmania nested Polymerase chain reaction

LPG = Lipophosphoglycin

LST = Leishmanin skin test

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LIST OF ABBREVIATIONS (CONTD.) Abbreviation Expanded version

MCL = Mucocutaneous Leishmaniasis

mM = Millimole

mg = Milligram

µgm = Microgram

ml = Millilitre

µl = Microlitre

µm = Micrometer

MIC = Minimum inhibitory concentration

M-CSF = Macrophage colony stimulating factor

MHC = Major Histocompetibility complex

MIF = Macrophage inhibitory factor

MgCl2 = Magnesium chloride

> = More than

medRNA = Mini exon derived RNA

MDR = Multi-drug resistant

n = Number

NNN = Novy McNeal Nicolle

NO = Nitric oxide

NK = Natural killer

ODC = Ornithine-decarboxylase

PCR = Polymerase chain reaction

PKDL = Post kala-azar dermal leishmaniasis

PCR-SHEL = PCR solution hybridisation enzyme linked assay

PCR-SSCP = PCR single stranded conformation polymorphisms

PAGE = Polyacrylamide gel electrophoresis

PBF = Peripheral Blood Film

% = Percent

RNA = Ribonucleic acid

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LIST OF ABBREVIATIONS (CONTD.)

Abbreviation Expanded version

r = Recombinant

SAG = Sodium antimony gluconate

Sb = Sodium stibogluconate

SD = Sodium deviation

SSU-rRNA = Small subunit ribosomal RNA

SDS = Sodium dodecyl sulphate

TNF = Tumour necrosis factor

TDR = Tropical disease research

Th = Helper T

TCR = T-cell receptor

TGF = Transforming growth factor

UV = Ultra violet

VL = Visceral Leishmaniasis

WHO = World Health Organization

Zn = Zinc

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SUMMARY

Diagnosis of kala-azar is problematic because of a variety of reasons. Definitive

diagnosis of Kala-azar requires tissue specimens, which are conventionally

obtained by organ needle aspiration for microscopic demonstration of amastigote

forms in stained smears. Organ aspiration and accurate examination of smears

also require technical skills that are not uniformly available in rural areas. PCR

testing of aspirate material improves parasitologic yield, but these methods are

seldom undertaken outside the research laboratories. Another interesting field for

definitive diagnosis of Kala-azar might be the detection of LD body from buffy

coat smear of peripheral blood. This cross-sectional study was conducted in the

Department of Microbiology, Mymensingh Medical College during the period

from July 2009 to June 2010. This study included 106 kala-azar cases were from

rural hospital (66.98%) and urban hospital (33.02%). Among them, 41 were

confirmed Kala-azar cases on the basis of LD body positive bone marrow in

microscopy. Another 65 Kala-azar cases were included on the basis of ICT

positive results for anti-Leishmanial antibody (rk39). The control group (n=44)

comprised of age and sex matched 44 healthy persons (Non kala-azar fever

patients) in the endemic area. Majority of the cases (58%) were in the age group

of 2-12 years. Male to female ratio of cases was 1.46:1. Most (68.87%) of the

Kala-azar cases were having earthen houses. All cases of Kala-azar were having

low-grade irregular fever with a mean duration of 3.77±1.32 months. Mean

enlargement of liver and spleen was found as 5.19±2.03 cm and 6.98±2.58 cm

respectively. Anaemia, leucopoenia, Thrombocytopenia were found in 34(83%),

18(44%), 33(80.48%) of confirmed cases of kala-azar respectively. Sensitivity

and specificity for buffy coat microscopy for LD body of confirmed Kala-azar

cases were found as (92.7%) and (95.45%) respectively. Sensitivity and

specificity of buffy coat PCR in confirmed kala-azar cases were 95.12% and

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100% respectively. Out of 65 ICT positive cases 92.30% yielded LD body

positive in buffy coat smear microscopy and 95.38% yielded PCR positive from

buffy coat. LD body detection in buffy coat smear became highly sensitive and

specific. Analyzing the findings of the present study, use of modified blood buffy

coat smear for confirmatory diagnosis of Kala-azar in endemic areas is

recommended. This study, also evaluated a prospective field for molecular

diagnosis of Kala-azar by an easy and accessible approach.

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CHAPTER 1

INTRODUCTION

The term leishmaniasis refers to various clinical syndromes caused by obligate

intracellular protozoa of the genus Leishmania. Sir William Leishman (British

Army Pathologist) reported about the parasite in 1903 and in the same year the

discovery was verified by Captain Charles Donavan (Professor of Physiology at

Madras University in India). Henceforth the parasite was named as the Leishman-

Donovan body or Leishmania donovani (Gibson, 1983).

Visceral leishmaniasis (VL) or Kala-azar is a re-emerging serious public health

problem in the Indian sub-continent targeting the poor (Joshi et al., 2008). Nearly

350 million people are at risk in 88 countries around the world. Currently an

estimated 12 million people are infected and around 2 million infections occur

each year (Rai et al., 2008). Of all the cases, 90% occur in India, Bangladesh,

Nepal, Sudan and Brazil (Ahasan et al., 2008). The Kala-azar cases estimation

was made for Bangladesh, India and Nepal and it shows that annual total number

of Kala-azar cases occurred from Bangladesh, India and Nepal are 136,500,

270,900 and 12,600 respectively (Joshi et al, 2008; Rijal et al, 2006).

The parasitic disease Kala-azar was first described in 1824, in Jessore district.

Bengal in what is now Bangladesh (Bern and Chowdhury, 2006). Previous

epidemiological studies show that 34 out of 64 districts of Bangladesh reported

kala-azar cases but 90% of them are from 10 districts. The cumulative reported

incidence of kala-azar by district from 1994 � 2004 shows that the worst hit area

in Bangladesh is Mymensingh followed by Pabna, Tangail, Jamalpur, Sirajganj,

Gazipur, Natore, Naogaon, Manikganj, Rajshahi and Naawabgaj (Rahman et al.,

2008) .

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In Mymensingh district, only 5 of 12 thanas reported kala-azar cases in recent

years. Using the population of the respective thana the denominator, the incidence

of kala-azar in Fulbaria thana ranged from 30 to 33/10,000/year since 2000, while

that in Trishal, the next most affected thana, ranged from 21 to 26/10,000/year.

Over the same period of time, the incidence in the other 3 endemic thanas,

Bhaluka, Muktagacha, and Goforgaon, ranged from 5 to 15 cases/10,000/year. For

the 5 endemic thanas of Mymensingh district, the mean annual reported incidence

since 2000 was 17/10,000/year, compared to 8.3/10,000 when the total district

population was used (Alam et al., 2009).

Kala-azar carries a high mortality rate ranging from 80% to 100% and even with

treatment, case fatality rates in excess of 10% are common (Salam et al., 2009).

Leishmaniasis development depends on several risk factors such as malnutrition,

immunosuppression, age, immunological status and genetic factors (Assimina et

al., 2008).

The risk of Kala-azar was highest for people in the 3- to 14-year and 15- to 45-

year age groups (Bern et al., 2005). Kala-azar mainly affects infants and young

children and male sex predominance (Uzair et al., 2004).

Kala-azar is typically caused by the Leishmania donovani complex, which

includes three species: L. donovani, Leishmania infantum, and Leishmania

chagasi. On the Indian subcontinent, the disease is almost exclusively caused by

L. donovani. The initial report of Leishmania tropica causing Kala-azar in India

was refuted by us and others. L. infantum is responsible for Kala-azar in children

in the Mediterranean basin (Begum et al., 2002).

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It is transmitted to man by phlebotomus (sandfly) in the Old World and

Lutzomiyia in the New World Sergentomyia is another vector found in

Baluchistan (Brito et al., 2000; EI-Hasan et al., 1995).

Occasional nonvector transmissions also have been reported through blood

transfusions, sexual intercourse, organ transplants, excrements of dogs, and

sporadically outside endemic areas. Congenital Kala-azar was described first in

1926 by Low and Cooke (Christoph et al., 1999). The incubation period is 3-6

months, though 10-24 years is also re-ported (Arias et al., 1996).

The disease is characterized by fever, hepatosplenomegaly, anaemia, leucopoenia

and hypergammaglobulinemia. Serious complications are cancrum oris,

dysentery, pneumonia, anaemia, agranulocytosis, jaundice, severe haemorrhage

and anasarca. Pulmonary tuberculosis may occur with Kala-azar (Cruz et al.,

2006).

The diagnosis of Kala-azar is complex because its clinical features are shared by a

host of other commonly occurring diseases, such as malaria, typhoid, and

tuberculosis; many of these diseases can be present along with Kala-azar (in cases

of coinfection); sequestration of the parasite in the spleen, bone marrow, or lymph

nodes further complicates this issue.

Laboratory diagnosis of leishmaniasis can be made by the following:

(i) demonstration of parasite in tissues of relevance by light microscopic

examination of the stained specimen, in vitro culture, or animal inoculation; (ii)

detection of parasite DNA in tissue samples; or (iii) immunodiagnosis by

detection of parasite antigen in tissue, blood, or urine samples, by detection of

nonspecific or specific antileishmanial antibodies (immunoglobulin), or by assay

for Leishmania-specific cell-mediated immunity (Sundar and Rai 2002).

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Definitive diagnosis of Kala-azar is made by demonstration of Leishmania

amastigotes in aspirates from lymph node, bone marrow or spleen. These

procedures have a sensitivity 58%, 60-85% and 96%, respectively (Zijlstra et al.,

1992). Direct demonstration of parasites in tissue smears, a technique that is

invasive and requires considerable expertise, by a �field test� (Boelaert et al.,

2007). Through splenic aspirate have high sensitivity, is associated with risk of

fatal hemorrhage in inexperienced hands (Kager et al., 1983). Marrow aspiration

either from illiac crest or from sternum is very much painful (Boelaert et al.,

1999; Chowdhury et al., 1993). In paediatric patients, detection rate of LD body

from bone marrow is higher because of higher parasitization (DGHS, 2009).

The Aspirates can also be used to culture leishmanian protozoas in NNN media

(1% defibrinated Rabbit blood + salt azar + Penicillin) and it takes about 1-3

weeks to grow. Other medias include, Grace�s culture media (Growth occurs

within 72 hours) and Sneader�s culture media. Culture is costly, time consuming

and requires sophisticated laboratory settings, so not a useful diagnostic tool

(Sundar and Rai, 2002).

The development of PCR has provided a powerful approach to the application of

molecular biology techniques to the diagnosis of leishmaniasis. Primers designed

to amplify conserved sequences found in minicircles of KDNA of leishmanias of

different species were tested in various tissues of relevance. In recent years PCR

based diagnostic methods with a wide range of sensitivities and specificities have

been described. The sensitivity of PCR with whole blood from Kala-azar patients

was 96%. PCR assay with buffy coat preparation to detect Leishmania was 10

times more sensitive than that with whole blood preparation and particularly good

results were obtained when proteinase K based methods were used. Proteinase K

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based PCR was able to detect 10 parasites/ml (Sundar and Rai 2002; Motazedian

et al., 2002; Schonioan et al., 2003).

The serodiagnosis of leishmaniasis (based on antibody detection) appears to be an

alternative to parasitological diagnosis. A number of serological techniques have

been developed for diagnosis of Kala-azar including ELISA, dot ELISA and

direct agglutination test. The sensitivity and specificity of such diagnostic

methods depends on the type, source and purity of antigen employed, as some of

Leishmania antigens have common cross-reactive epitopes shared with other

microorganisms (Sarkari et al., 2005). rk39 ICT strip test is easy to perform at

field. It shows high sensitivity and specificity (Salam et al., 2009; Bern et al.,

2000).

Antigen detection is more specific than antibody-based immunodiagnostic tests. A

new latex agglutination test (KATEX) for detecting leishmanial antigen in urine

of patients with Kala-azar has showed sensitivities between 68 and 100% and a

specificity of 100% in preliminary trials. The antigen is detected quite early

during the infection and the results of animal experiments suggest that the amount

of detectable antigen tends to decline rapidly following chemotherapy. The test

performed better than any of the serological tests when compared to microscopy.

Large field trials are under way to evaluate its utility for the diagnosis and

prognosis of Kala-azar (Attar et al., 2001; Fakhar et al., 2006).

Another interesting field for definitive diagnosis of Kala-azar might be the

detection of LD body from buffy coat smear of peripheral blood. Because, when

blood is settled in presence of an anticoagulant, the WBCs are deposited and

concentrated in buffy coat at the interface of RBCs and plasma. If buffy coat is

considered for making a smear, it is supposed to show high content of WBCs

including monocytes. The monocytes parasitized by LD bodies, if present, will be

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higher in the smear. Thus the rate of LD body positivity is expected to be higher

(Shamsuzzaman et al., 2007).

Considering the background described above definitive diagnosis of kala-azar is

technically difficult job. LD body from blood buffy coat is an easy and

noninvasive approach for definitive diagnosis of Kala-azar. The present study

explored the reliability and effectiveness of LD body detection in blood buffy coat

smear and also to identify Leishmania donovani kinetoplast DNA from blood

buffy coat of Kala-azar patients by Nested PCR (Ln-PCR) which is non-invasive,

reliable, sensitive and specific. Therefore the present study was designed with the

following objectives.

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OBJECTIVES

General objective: To evaluate buffy coat- a noninvasive method for definitive

diagnosis of kala-azar.

Specific Objectives

i. To diagnose kala-azar by detection of LD bodies from bone marrow

aspiration in clinically suspected cases.

ii. To evaluate buffy coat smear for detection of LD bodies in clinically

suspected cases.

iii. To identify Leishmania donovani kinetoplast DNA from blood buffy

coat of Kala-azar patients by Nested PCR (Ln-PCR)

iv. To detect anti rK39 antibody by ICT in Kala-azar patients

v. To determine the sensitivity and specificity of buffy coat smear for

diagnosis of kala-azar

vi. To find out haematological changes in kala-azar patients

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CHAPTER 2

REVIEW OF LITERATURE

2.1 Historical background

The organism of kala-azar was first described in 1903 by Sir William Leishman

who examined the spleen of a British soldier stationed at Dumdum near Calcutta,

India. Later the same year, Charles Donovan verified this finding and the

organism in often called Leishman-Donovan (LD) body (Chulay, 1991). The

genus Leishmania was created by Ross in 1903 to include Leishmania donovani.

In India, the disease is known a kala-azar, meaning �black sickness or fever� as

the disease turns the color (pigmentation) of the skin black, the word �kala� means

�black and �azar� means �deadly�, thereby signifying a fatal illness (Chatterjee,

1982). In 1904, Roger observed the conversion of amastigotes of promastigotes in

culture (Chulay, 1991). A similar parasite was observed in a disease of children in

the Mediterranean countries by Nicolle in 1908, proposed the name of infantile

kala-azar for this disease and designated the parasite as L. infantum (Chatterjee,

1982). Further investigation revealed that it was a strain of L. donovani. The

parasite of South American Kala-azar originally named as L. chagasi in 1937, was

also found to be identical of L. donovani. Promastigotes were found in sandflies

by Alder and Theodor in 1925 (Chulay, 1991). In 1940, it was demonstrated the

Plebotomus argentipes was the vector of Indian kala-azar (Birley, 1993) and in

1942, in India kala-azar was transmitted experimentally to human volunteer by

sandfly bite (Swaminathan et al., 1942).

Irregular epidemic waves have swept through Assam, Bengal and Bihar since the

1800s with a frequency of 15-20 years. In 1890-1900 an epidemic swept Assam

which depopulated whole villages and reduced populations over large areas. In

1917 another epidemic started in Assam and Bengal and reached its height about

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1925 and mysteriously subsided, until by 1931, it was almost gone. In 1937 a new

outbreak began in Bihar (Birley, 1993). Before the Second World War, kala-azar

was endemic in Assam, Bengal (a part of which is now Bangladesh), Bihar and

some other parts of Indian subcontinent. Due to insecticide spraying as a part of

malaria eradication campaign in 1958-1964, kala-azar almost disappeared from

chasten states of Indian and Bangladesh but with the cessation of insecticide

spraying there has been a resurgence of Kala-azar in these regions (Thakur et al.,

1981; Rahman and Islam, 1983).

2.2 World�s situation of Leishmaniasis

Kala-azar is endemic in the tropical and sub-tropical regions of Africa, Asia, the

Mediterranean, Southern Europe, South and Central America. The distribution of

Kala-azar in these areas however is not uniform; it is patchy and often associated

with areas of drought, famine and densely populated villages with little or no

sanitation (WHO, 1991; Rab and Evans, 1995).

In Pakistan 239 cases of Kala-azar due L. infantum were reported between 1985

and 1995. Off those, 52% were children below the age of 2 years and 86% were

below 5 years. This figure represented an increased of ten-fold in infantile Kala-

azar cases over the 10 year period from 0.2 to 2 per 100 000 population and male

cases out numbered female cases by three times (Rab and Evans, 1995). Kala-azar

has been known to exist in the Himalayas in Pakistan for over three decades.

However recently sporadic cases are beginning to appear in the North West

Frontier Province, Punjab and Azad Jammu and Kashmir. All of these areas are

mountainous and contain large farming communities (Rab and Evans, 1995).

In neighbouring India Kala-azar is endemic in the states of Bihar, Uttar Pradesh

and West Bengal. One of the largest epidemics occurred in 1978 in North Bihar

23

were over half a million people fell victim to Kala-azar. In the first eight months

of 1982, 7500 cases were reported in India and in one year alone between 1987

and 1988, 22 000 cases of Kala-azar were registered (WHO, 1991). In Bangladesh

cases of Kala-azar greatly declined between 1953-1970, probably as a result of

mass chemotherapy with pentavalent antimonials and wide spread spraying with

DDT to control malaria. Following the end of the malaria control programme in

1970, sandfly vector populations increased and so did the cases of Kala-azar and

currently appear at a rate in excess of 15000 per year (Al-Masum et al., 1995). In

Brazil, Kala-azar is distributed widely in the south, east and the central regions of

the country. Kala-azar commonly affects poor and malnourished children below

the age of 15 years (Arias et al., 1996). The disease is highly endemic in the states

of Bahia and Ceara, which together account for 70% of the total cases of Kala-

azar in Brazil, upto 1989, 15000 cases of Kala-azar was recorded in multiple

states (WHO, 1991). Recently the foci of Kala-azar has shifted from rural villages

to large cities probably as a result of migration of settlers from villages into these

cities creating dense population and living in sub-standard house with improper

sanitation and keeping farm animals in their gardens. The two cities of Teresin

and Sao Luis together accounted for 40-50% of the total number of Kala-azar

cases. During 1993 and 1994, approximately 3000 cases per year were recorded

(Arias et al., 1996). In Central America, the disease has been increasing in Costa

Rica, Honduras and Nicaragua. This is most probably due to an increase in the

human population and their movements in and out of these areas (Carreira, 1995).

First case of Kala-azar in Sudan was reported in 1938.

Since then the disease has become widespread and endemic in south and eastern

parts of the White Nile and Upper Nile states (Hashim et al., 1995). Other

sporadic areas include the provinces of Kasala, Jonglei and Kapoeta in the south,

E1 Fasher an E1 Nahud in the west and also north of Khartoum (WHO, 1991). In

24

most countries, males are almost twice more likely to be Kala-azar than females,

with young children being at the highest risk. In the village of Um-Salala in

eastern Sudan, the average age of Kala-azar patients was found to be 6.6 years

with a male to female ratio of 1.8:1. Annual incidence was recorded as 38.4 per

1000 population between 1991 and 1992 (Zijlstra et al., 1994). The first case of

Kala-azar in Ethiopia was documented in 1942 in the southern parts of the

country. Since then the disease has spread to become endemic in the Segen, Woito

and Gelana river valleys. The highest incidence was recorded in Aba Roba area

(WHO, 1991). It was found that 58% of the people affected were children below

15 years of age with the lowest risk groups being males above the age of 39 years

and females above 24 years (Ali and Ashford, 1994). In Somalia, Kala-azar is also

endemic with higher prevalence among children below the age of 15 years. Males

are three times more susceptible than females (Shiddo et al., 1995).

2.2.1 Epidemiology

Kala-azar is a re-emerging serious public health problem in the Indian sub-

continent targeting the poor (Joshi et al., 2008). Nearly 350 million people are at

risk in 88 countries around the world. Currently an estimated 12 million people

are infected and around 2 million infections occur each year (Rai et al., 2008). Of

all the cases, 90% occur in India, Bangladesh, Nepal, Sudan and Brazil (Ahasan et

al., 2008).

The parasitic disease Kala-azar was first described in 1824, in Jessore district.

Bengal in what is now Bangladesh (Bern and Chowdhury, 2006). Previous

epidemiological studies show that 34 out of 64 districts of Bangladesh reported

kala-azar cases but 90% of them are from 10 districts. The cumulative reported

incidence of kala-azar by district from 1994 � 2004 shows that the worst hit area

in Bangladesh is Mymensingh followed by Pabna, Tangail, Jamalpur, Sirajganj,

25

Gazipur, Natore, Naogaon, Manikganj, Rajshahi and Naawabgaj (Ahasan et al.,

2008) .

In Mymensingh district, only 5 of 12 thanas reported kala-azar cases in recent

years. Using the population of the respective thana the denominator, the incidence

of kala-azar in Fulbaria thana ranged from 30 to 33/10,000/year since 2000, while

that in Trishal, the next most affected thana, ranged from 21 to 26/10,000/year.

Over the same period of time, the incidence in the other 3 endemic thanas,

Bhaluka, Muktagacha, and Goforgaon, ranged from 5 to 15 cases/10,000/year. For

the 5 endemic thanas of Mymensingh district, the mean annual reported incidence

since 2000 was 17/10,000/year, compared to 8.3/10,000 when the total district

population was used (Bern and Chowdhury, 2006).

The resurgence of kala-azar was associated with lack of spraying which allowed

the build-up of sand fly population. It also revealed that about 90% of cases of

kala-azar came from low-income group who were living in the houses plastered

with mud and cow-dung. It was also noted that the disease were low or rare in

malaria endemic areas where regular DDT spraying was going on (Masum et al.,

1991).

2.2.2 Mode of Transmission

Transmission of kala-azar is caused by the bite of infected female sandfly of the

genera Phlebotomus and Lutzomyia. Some of the species feed on man and also a

variety of warm and cold-blooded animals, which is an important factor in

spreading the disease. A sandfly become infected 14-18 days after the ingestion of

the infected blood meal and remains infected throughout its lifetime and are

capable of infecting several persons (Cheesbrough, 1999).

26

Transmission of Kala-azar may take place by contamination of bite wound or

contact when the insect is crushed during the time of biting (Park and Park, 2000).

Accidental inoculation of parasites during laboratory work may result in

leishmaniasis. Transmission by blood transfusion has also been recorded.

Congenital infection from an infected mother may occur rarely. Transmission of

infection to fetus in utero may occur due to placental defect (Bahr and Bell, 1987).

Transmission during coitus was also recorded (Chatterjee, 1980).

A case of fatal leishmaniasis was also noted in renal transplant patient due to

kidney transplantation (Vanorshovan et al., 1979).

2.2.3 Vectors

Sandfly species and subspecies of Phlebotomus in the Old World (Asia, Africa

and Europe) and Lutzomyia in the New World (Central and South America) are

the only proven vectors of Leishmania (WHO, 1990).

In the Central and South America Lutzomyia longipalpis is the main vector for

transmission of Kala-azar. In the Mediterranean region, Phlebotomus perniciousus

and Phlebotomus ariasi are important vectors while in China Phebotomus

chinensis and Phlebotomus alexandri are the proven vectors. In East Africa

including Sudan Phlebotomus martini and Phlebotomus orientalis are considered

as vectors for the diseases. In India, for the anthropologic form of Kala-azar the

proven vector is Phlebotomus argentipes (WHO, 1990; Swaminathan et al.,

1942). Besides these many other species have been implicated as vectors of

leishmaniasis.

In Bangladesh, during an outbreak of kala-azar at Kalihati Thana of Tangail

districts sandfly species of Phlebotomus argentipis, Segentomyia babu babu,

Sergentomyia barrudi and Sergentomyia shortill were identified (Masum et al.,

27

1990a). The sandflies collected from Thakurgaon district of Bangladesh during an

outbreak of Kala-azar were identified as Phlebotomus argentipes, Phlebotomus

papatasi, Sergentomyia babu babu and Sergentomyia barrudi (Masum et al.,

1990b).

An entomological study on vector of kala-azar in Shahjadpur thana of Sirajgonj

district showed that the species were Phlebotomus argentipes, Phlebotomus

malabaricus and Phlebotomus minutus. Among these Phlebotomus argentipes

was 70.6% (Ahmed and Ahmed, 1983).

In Bangladesh on effort so far has been made to implicate sandflies as vector(s) of

leishmaniasis, however, Phlebotomus argentipes is a recognized vector of kala-

azar in India and in Bangladesh this species occurs in areas where cases of kala-

azar have been found (Elias et al., 1989).

2.2.4 Reservoirs

Broadly speaking, there are two types of Kala-azar, namely zoonotic and

anthroponotic Kala-azar (WHO, 1990). In the anthroponotic form, human, act as

reservoir and in the zoonotic form animals are the reservoir.

Human- In the anthroponotic form, which is prevalent in India, Bangladesh and

some countries of East Africa, humans are directly involved as reservoir (WHO,

1990). All efforts to find a zoonotic reservoir in Indian Kala-azar has so far been a

failure (Srivastava and Chakravarty, 1984). Since PKDL may persist for up to 20

years such patients may act as a chronic reservoir of infection (Chulay, 1991) and

an outbreak of Kala-azar has been traced to case of PKDL (Addy and Nandy,

1992).

28

Dogs- In the zoonotic form, dogs are the principal reservoir in Europe and Africa

around the Mediterranean regions. Dogs are also important reservoirs in China

and South and Central America.

Wild canines- In southern France and central Italy foxes with inapparent

infection are the reservoir. Foxes are also reservoir in Brazil and jackals are

probably an important source of sporadic mainly rural cases that occur in the

Middle East and Central Asia.

Rodents and others- Rattus rattus, the common peridomestic rat in many

countries, has been found to be infected with various Leishmania species in both

Old World and New World. While its role in the maintenance of parasite

populations has not been fully established, it is strongly suspected as a secondary

reservoir host of L. infantum in Italy (WHO, 1990). L. donavani has been isolated

from Arvicanthis nilotica and other rodents in Sudan and rodents are probably

important in maintaining enzoonotic foci in interepidemic period.

2.3 Taxonomy of Leishmania (WHO, 1990)

Order : Kinetoplastida

Family : Trypansomatidae

Genus : Crithidia, Leptomanas, Herpetomonas, Blastocrithidia,

Leishmania, Sauroleishmania, Trypanosoma, Phytomonas

and Endotrypamm

Subgenus : Leishmania, Viannia

Complex : L. donovani, L. tropical. L. major, L. aethiopica, L. mexicana,

L. braziliensis, L. guyanensis.

Species : L. archibaldi, L. killicki, L. major, L. aethiopica, L.

amazonensis, L. braziliensis, L. guyanensis, L. panamensis, L.

panamensis, L. changasi, L. tropica, L. garnhami, L.

Peruviana, L. donovani, L. mexicana, L. infantum, L. pifanoi,

L. arabica, L. venezuelensis, L. gerbilli, L. enriettii.

29

(The classification of genera and subgenera is based on extrinsic characters and

that of the complexes mainly on intrinsic characters-isoenzymes).

2.4 Aetiology

Causative Agents

Seven complexes consisting of 17 Leishmania species i.e. Leishmania donovani

complex, Leishmania tropica complex, Leishmania major complex, Leishmania

aethiopica complex, Leishmania mexicana complex, Leishmania braziliensis

complex and Lcishmania guyanensis complex have been identified as causative

agents of leishmaniasis all over the world (WHO, 1990).

Kala-azar is caused by parasite species of the Leishmania donovani complex

which include Leishmania donovani donovani, Leishmania donovani infantum

and Leishmania donovani chagasi (in this thesis the three species has been

referred to as L. donovani, L. infantun and L. chagasi respectively). Some

undefined or unspecified species belonging to the L. donovani complex has been

isolated from Kala-azar patients in Kenya, Ethiopia and Somalia (Pearson and

Sousa, 1990).

Beside these species mentioned above, other species generally associated with

cutaneous leishmaniasis have been isolated from Kala-azar patients in different

regions of the world (Sacks et al., 1995) isolated L. tropica from patient with

classical Indian Kala-azar. L. major has been isolated from a patient of Kala-azar

in Israel (WHO, 1990). Kala-azar caused by L. amazoniensis has been reported

from Bahia state in Brazil (Pearson and Sousa, 1996). Viscerotropic syndrome

caused by L. tropica affected a small number of American troops during

Operation Desert Storm (Magill et al., 1993).

30

Old World, anthroponotic Kala-azar is caused by L. donovani. In India,

Bangladesh, Nepal parts of China and East Africa Kala-azar is believed to be

caused by L. donovani but other species has been identified as mentioned above.

Zoonotic Kala-azar in Mediterranean region, China, Middle East and parts of Sub-

Saharan Africa is caused by L. infantum with dog being the principal reservoir.

New World zoonotic Kala-azar is caused by L. chagasi and L. infantum, here as

well, dogs are the main reservoir.

2.5 Morphological Forms

Leishmania are digenetic (existing two forms) protozoa which exists as:

i. Amastigote form and

ii. Promastigote form.

Staining characteristics- Romanowsky dyes stains chromatin of the nucleus and

nucleic acid containing kinetoplast a brilliant red or violet, whereas the cytoplasm

is stained pale blue (Neva and Sacks, 1990).

With Leishman�s stain the cytoplasm appears blue, the nucleus pink or violet and

the kinetoplast, bright red (Chatterjee, 1982).

i. Amastigote form- Amastigote are aflagellar, obligate intracellular form

which reside within mononuclear phagocytes of their vertebrate hosts

including man. Amastigotes appear as round or oval bodies ranging

from 2-3 m in major diameter. The size of amastigote from different

species is known to vary. The cytoplasm of the amastigote often stain

the same as the host cell cytoplasm and only the nucleus and kinetoplast

distinguished. The nucleus of the parasite occupies a central position or

along side of the cell membrane. The kinetoplast lies adjacent to the

nucleus either tangentially or right-angle to it. The kinetoplast stains

more densely than the nucleus and it is variable in shape being round,

31

rod-shaped or curved in profile (Chaterjee, 1982; Neva and Sacks,

1990).

ii. Promastigote form- Promastigotes are flagellated extracellular forms

which are found in the gut of sandflies and in vitro culture. The

flagellar promastigote from measure 10-20 m in length not including

the length of the flagellum which may equal the body length. The pale

blue staining cytoplasm contains a centrally placed nucleus. The kin

kinetoplast lies about 2 m from the anterior end and the flagellum

emerges anteriorly. The overall shape is that of a spindle with the

posterior end gradually tapering to a point (Neva and Sacks, 1990).

2.6 Life-Cycle

In sandfly host- The amastigotes are ingested with the first blood meal of the

female sandfly in which they become transformed almost immediately into

promastigotes which multiply in the midgut and then migrate forwards to the

anterior part of the thoracic midgut or �cardia�. From here they move forward to

contaminate the mouth parts to be regurgitated into the wound caused by the bite

at the second blood meal. In all cases the infection is transmitted by bite.

Development in the sandfly from amastigotes to infective promastigote stage

(metacyclic promastigotes) varies from 5-10 days (Manson-Bahr and Bell, 1991).

In mammalian host- After inoculation by the sandfly, either into a capillary or

the dermal tissue, the promastigotes encounter macrophages which actively search

them out and phagocytose them by receptor-medicated endocytosis. The

promastigote changes into amastigotes in a phagolysosome where it multiplies by

binnary fission. Multiplication goes on continuously till the cell becomes packed

with parasites. The host cell in thereby enlarged and eventually ruptures or the

amastigotes leave the macrophages by penetrating the cell membrane. The

amastigotes thus released into the circulation are again either taken up by, or

32

invade fresh macrophages and the cycle is repeated. In this way the entire

reticuloendothelial system becomes progressively infected. In the blood stream,

some of the free amastigote are phagocytosed by the neutrophilic granulocytes

and monocytes (macrophages). A blood-sucking sandfly draws these free

amastigote forms as well as those within the monocytes during its blood-meal

(Chatterjee, 1982; Manson-Bahr and Bell, 1991).

2.7 Determinants of virulence

2.7.1 Surface Lipophosphoglycan (LPG)

Lipophosphoglycan (LPG) are heterogenous molecules and constitute the most

abundant surface component; each call has more then 106 copies, LPG is present

in both leishmanial stages and is released as �excretory factors�. The molecular

structure of LPG consists of three portions; terminal repeats of phosphorylated

saccharides, a phosphorylated heptasaccharide core and lyso-phosphotodylinositol

(Turco et al., 1987). The terminal saccharides repeating units are also structurally

unique and vary with Leishmania species. The difference in this portion of the

LPG distinguishes antigenically different Leishmania species. Antigenic

heterogeneity is further contributed to, probably even more significantly, by

another group of related surface inositol glycolipids. The tissue tropism of

different Leishmania species may be related to the variations in their surface

glycolipids (Handaman et al., 1987).

LPG and related glycolipids play important biological roles in Leishmania

macrophage interactions. The receptor for LPG appear to be complement receptor

(CR) 3 and p150, 95 (CR4) (Chang et al., 1990). In addition, LPG enhances the

survival of promastigotes in macrophages (Handman et al., 1986). Experimental

evidence suggests that the mechanism of this protection may be based on the

action of LPG to scavenge oxygen free radicals (Chan et al., 1989) and or to

33

inhibit relevant enzymes, e.g. liposomal glycosidase and protein kinase C. Other

proposed biological action of LPG includes inhibition of lymphoproliferative

response and activation of T suppressor cells (Chang et al., 1990). Leishmanial

virulence has been related to quantities and qualitative changes of promastigote

LPG.

The Leishmania LPG and related glycolipids are highly immunogenic owing to

their unique structural features (Tolson et al., 1989). They have long been

exploited as excretory factors for serotyping of Leishmania species (Turco, 1988).

2.7.2 Glycoprotein (gp) 63

Surface glycoprotein known as gp63 was initially recognized as a major surface

antigen of promastigotes by using monoclonal antibodies and surface radio

iodination of living cells (Chang et al., 1990). It constitutes about1% of the total

cellular proteins or 500,000 copies per cell (Bordier, 1987; Bouvier, et al., 1985).

The gp63 protein is seen to migrate on SDS polyacrylamide gels between 60-65

kD depending on the species of Leishmania (Wilson et al., 1989) and which is the

major antigenic protein of most promastigotes (Colomer-Gould et al., 1985). Gp

63 has been found in all major pathogenic Leishmania species (Bourver et al.,

1987), and in both stages (Colomer-Gould et al., 1985) endowed with proteolytic

activity (Etges et al., 1986). Gp63 has been shown to cleave C3 into C3b and

other C3 products (Chaudhuri and Chang, 1988).

By virtue of its proteolytic activity, universal presence, abundance and surface

localization, gp63 is thought to be a Leishmania virulence factor. The abundance

of gp63 is often correlated with infectivity (Kweider et al., 1989; Wilson et al.,

1989) and host-parasite interactions (Russell et al., 1986; Wilson et al., 1988).

Because of gp63�s proteolytic activity, it may function in the CR-mediated

34

endocytosis of promastigote (Da Silva et al., 1989) and protection of Leishmania

from intralysosomal microbicidal factor (Chang et al., 1990). Leishmania gp63 is

immunogenic; anti-gP63 antibodies have been reported in sera from patients with

leishmaniasis (Heath et al., 1987; Reed et al., 1987; Murray, et al., 1989). Gp63 is

potentially useful antigens for immunodiagnosis (Reed et al., 1987) and

immunoprophylaxis (Yang et al., 1990).

2.7.3 Acid phosphatases

The cell surface acid phosphatases (ACP) was initially discovered on the

promastigote stage using ultrastructure cytochemistry. Two forms of ACP

namely, membrane-bound ACP and secretary ACP, are present. The two forms

are antigenically distinct and each probably exists as multiple isoenzyme present

in both stages of most Leishmania species. One form of the membrane-bound

ACP and the secretory ACP have been purified from L. donovani and found to be

homodimers of 120 kD and 134 kD (Chang et al, 1990). All these different forms

of ACP are nonspecific monoesterase capable of hydrolyzing a variety of

phosphorylated substrates.

The membrane-bound ACP purified from promastigotes reduced the respiratory

burst of neutrophils (Remaley et al., 1985) and is itself resistant to oxidative

metabolites (Saha et al., 1985). In addition, it has been shown to dephosphorylate

certain phospholipids and phosphoroteins. Thus, the ectoenzyme is thought to

protect Leishmania species by interfering with the regulatory mechanism of the

macrophages that produces microbial free radicals (Glew et al., 1988). Its

production in large quantity elicits humoral immune response of the host and may

conceivably to the pathobiology in leishmaniasis (Chang et al., 1990).

35

2.7.4 Nucleotidases

5� nucleotidase and 3� nucleotidase/nuclease have been reported to exist on the

surface of some trypanosomatid protozoa including Leishmania species. The 5�

nucleotidase has an electrophoretic mobility of about 70 kD on SDS-PAGE and is

active in degrading both ribo-and deoxyribonucleotides. The 3� nucleotidase

/nuclease of 43 kD is more abundant. The latter enzyme is most active with 3�

AMP and prefers RNA over DNA as substrate. The alkaline pH optima of both

enzymes suggest that they probably serve such functions for promastigotes in the

sandfly gut better than for amastigotes in the phagolysosomes of the macrophages

(Chang et al., 1990).

2.7.5 Transporters

The plasma membrane of Leishmania has been shown biochemically and

genetically to possess the transport systems for folate, glucose, nucleosides,

proline and ribose. They also possess a cation or proton transporting ATPase,

which is apparently crucial for the homeostasis of Leishmania species and the

transport of nutrients necessary for their adaptation to the changing environment

in their life-cycle (Chang et al., 1990).

2.7.6 Cystenic proteinase and megasomes

The cysteine proteinase, a conserved and wide-spread enzyme, has been found in

Leishmania species generally associated with cutaneous form of leishmaniasis of

South American origin. It is present in an unusual organelle, or megasome a

modified lysosome, which is noticeable in the promastigotes grown to stationary

phase and becomes fully developed as they differentiable into amastigotes. This

enzyme is proposed to sever a degenerative role, possibly for the nutritional

benefits of the amastigotes and for releasing ammonia or other amines to

36

modulate that host lysosomal activity for the parasites� intracellular survival and

is functionally important to amastigotes (Chang et al., 1990).

2.7.7 Heat-shock proteins

Exposure of promastigotes to elevated temperatures results in an over-expression

of the classic heat-shock genes other genes. Multiple protein bands emerge shortly

after heat-shock and their number varies with different species. Only one of these

proteins has been positively identified immunologically as equivalent to Hsp70

protein. Most intruging is the increase in the virulence seen with briefly heat-

shocked promastigotes. What molecular changes in these promastigotes account

for virulence remains uncertain (Chang et al., 1990).

2.7 Pathogenesis

Despite the wide range of variation of the geographical distribution, clinical

manifestations and species involved, leishmanial infection shares a common

feature, namely the parasitizaiton of the phagocytic cells of major organs of the

reticuloendothelial (RE) system such as spleen, liver, blood, bone morrow and

skin (WHO, 1990).

With the bites of infected sandfly, the metacyclic (infective) forms of parasite are

inoculated into the microwound of the skin. Here the promastigotes are exposed to

IgG and IgM which opsonize them and are killed by activation the membrane

attach complex of complement through the classical pathway (Pearson and

Steigbige, 1980). Those escaping the lethal effect of serum bin to macrophages.

Lipophosphoglycan 9LPG) and gp63 has been considered parasite ligands

(Handman and Goding, 1985; Rizvi et al., 1988; Russell and Wright, 1988), with

complement receptor CR 1 and CR 3 and mannose-fucose receptor as the

corresponding macrophage receptors (Blackwell et al., 1985; Mosser and Edelson,

1985; Da Silva et al., 1989). After receptor-mediated endocytosis the parasite

37

resides within the phagolysosomes, thus are sheltered from the body�s immune

system. Side by side, they overcome the microbicidal conditions of the

macrophage phagolysosomes. Amastigotes and metacyclic promastigotes appear

to evade oxygen-dependent destruction by triggering a minimal respiratory burst

during infection due to the use of C3 receptors for internalization (Da Silva et al.,

1989). LPG scavenges oxygen free radicals (Chan et al., 1989) and inhibits

relevant enzymes e.g. lysosomal glycosidase and protein kinase C which may

enhance the survival of promastigotes in macrophages (Chang et al., 1990). Gp63,

a parasite ectoenzyme, has been shown to protect lipid-protein substrates from

intralysosomal degradation within macrophages. Protection against low pH that

exists within the phagolysosmes, is accomplished by the action of a membrane

proton translocating ATPase which is located on the cytoplasmic side of the

parasite surface membrane and acts by coupling ATP hydrolysis to proton-

pumping activity (Chang et al., 1990).

The parasites multiply by binary fission within the phagolysosomes. The infected

macrophages secrete colony-stimulating factors which stimulate precursor cells of

macrophages thereby providing new target cells for the parasites and form a

granuloma with epitheliod cells and giant�s cells in the dermatotropic species and

hyperplasia of the reticuloendothelial cells in the viscerotropic species (WHO,

1990). In Kala-azar, the major host response is cellular and the amastigotes in

macrophages are killed due to increased production of oxygen and nitric oxide

metabolites in the macrophages (Liew et al., 1990) stimulated by lymphokines

particularly interferon- (IFN-), from activated T-helper 1 cells generated during

the immune response. The released amastigotes are destroyed extracellularly with

the appearance of delayed type hypersensitivity. So, in majority cases, the

infections are mild and self-limiting (WHO, 1990). A small fraction of individuals

who develop specific suppression of cell-mediated immunity permits the

38

dissemination and uncontrolled multiplication of parasites leading to disease and

complications.

2.9 Immunology of leishmaniasis

2.9.1 Natural Immunity

There is considerable variation in the immune response to infection in Kala-azar.

Some animals are completely resistant to infection and disease in man is very

variable. Resistance to infection with Leishmania donovani subspecies infection

in mice has been shown to be under genetic control. Infection with all forms of

leishmaniasis which is allowed to run its natural course immunizes against

reinfection with the homologous strain. Once recovered from infection either

naturally or by chemotherapy, established immunity is life long and their are no

reliable records of second attack of Kala-azar (Manson-Bahr and Bell, 1991).

2.9.2 Acquired immunity

In Kala-azar there is a wide spectrum of disease varying from self-healing and

subclinical infection to overt kala-azar syndrome. The majority of infection is

self-healing and subclinical. In a study in Italy it was found that clinical illness

developed in only 3% of those infected (Pampiglione et al. 1975), while others

suggested that upto 85% of infected individuals may spontaneously control

infection (Evans et al. 1992; Holaday et al. 1993).

Infection with subspecies of Leishmania donovani complex is associated with a

profound impaired cellular response but marked humoral response. Kala-azar

patients fail to respond to L. donovani antigens in term of delayed type

hypersensitivity (Rezai et al., 1978; Ho et al., 1983; Halder et al., 1983)

lymphocyte proliferation in response to both mitogens (Ghose et al., 1979) and

parasite antigens (Halder et al., 1983) and IL-2 and IFN- production in vitro and

39

in vivo (Carvalho et al., 1985). However, these immune responses are restored

after successful chemotherapy (Manson-Bahr and Bell, 1991).

2.9.3 Genetic regulation of Leishmaniasis

Several different host genes control the infection of distinct species of

Leishmania. In the murine model, two stages of genetic regulation have been

identified-primary innate susceptibility or resistance and subsequent acquired

immunity. This division is particularly clear in Kala-azar where innate

susceptibility or resistance is controlled by expression of a single autosomal

dominant gene locus designated Lsh/Ity/Bcg (Bradley, 1977; Plant et al., 1982).

Natural resistance-associated macrophage protein gene 1 (Nramp1) which had

been isolated as a candidate gene has been established as an allele of Lsh/Ity/Bcg

(Formica et al., 1994). Acquired immunity is controlled by at least three

additional genes Rld-1 (lined to major his compatibility loci, H-2), H-11 linked

gene and Ir-2 (Leiw and O�Donnell, 1993).

As yet little is known about the effect of genetic variation on leishmaniasis in

humans and genetic determinants of human leishmaniasis have yet to be

elucidated. However both visceral and cutaneous leishmaniasis can exhibit a wide

clinical spectrum in human. In mucocutaneous leishmaniasis caused by L.

braziliensis, sign of genetic variation have been recorded (Walton and Valverde,

1979).

2.9.4 Humoral immune response

A marked hypergammaglobulinemia and absence of detectable cell-mediated

immunity are the principal immunological features of Kala-azar. There is

significant increase in IgG and IgM levels but not of IgA levels (Aikat et al.,

1979; Galvao-Castro et al., 1984). The rise in IgM level is less than that of IgG.

40

Elevated level of IgE was also detected in sera of Kala-azar patients (Peleman et

al., 1989).

Both Leishmania-specific and non-specific antibodies are produced during

infection with Leishmania species. Although both IgM and IgG antileishmanial

antibodies were detected in sera from Kala-azar patients, IgG titre higher than

those of IgM. No correlation was found between the titre of IgG or IgM

antileishmanial antibodies and the total serum levels of IgG or IgM (Galvao-

Castro et al., 1984). By immunoblot analysis Leishmania specific antibodies in

the IgG and IgM classes were observed but there was no reactivity in the IgA and

IgE classes (Rolland-Burger et al., 1991).

Non-specific antibodies reacting to haptens, foreign proteins and autoantigens

have been detected in sera of Kala-azar patients. Autoantibodies to IgG, smooth

muscles and single-stranded DNA was observed in many instances (Aikat et al.,

1979; Galva-Castro et al., 1984).

The marked increase of immunoglobulin levels in patients with Kala-azar and

demonstration of variety of antibodies against proteins unrelated to parasite and

against autoantigens is suggestive of polyclonal B cell activation (Weintraub et

al., 1982; Campos Neto and Bunn-Moreno, 1982). The mechanisms that may

induce polyclonal B cell activation are not completely understood but it has been

demonstrated that parasite-derived mitogens from promastigotes induce

polyclonal B cell activation in animal�s activation (Weintraub et al., 1982;

Campos Neto and Bunn-Moreno, 1982). Activated T cells were demonstrated

in Kala-azar patients which secrete cytokines involved in B cell growth and

differentiation and thus may contribute to the polyclonal B cell activation

(Raziuddin et al., 1992).

41

Antileishmanial antibodies have been shown in vitro to lyses promastigotes in

presence of complements (Pearson and Steigbigel, 1980; Mosser and Edelson,

1984) to promote phagocytosis (Herman, 1980). However there is little evidence

for a corresponding role in-vivo for antibody in determining the outcome of

leishmanial infection. Available evidence argues strongly against a protective role

in controlling leishmaniasis.

The increased level of Leishmania-specific and non-specific antibodies has been

exploited in the various serological diagnosis of Kala-azar.

Hypergammaglobulinemia has formed the basis of aldehydes or formol gel test

used for identifying patients of Kala-azar. Cross-reactive antibodies gives rise to

positive results in complement fixation test using mycobacterial antigens.

Leishmania-specific antibodies are detected by ELISA, IFAT and DAT using

either whole or whole cell lysate as antigens and are used for specific diagnosis of

leishmaniasis. In polypeptides are detected for the purpose of diagnosis of

leishmaniasis.

2.9.5 Cell-mediated immune response

Leishmaniasis has emerged as a model system for study of T-cell mediated

immunity. Several cell types may have a role in cell-mediated immune response

in leishmaniasis but the principal role is played by CD4+ T helper cells.

Natural killer (NK) cells

Little work has been done on the possible role of NK cells in leishmaniasis. It has

been shown that mice profoundly deficient in NK cell activity are modestly

capable of eliminating L. donovani infection than the normal controls. Therefore

NK cells may be involved in the response to Kala-azar. It has been shown that

early presence of IFN- favors CD4+ Th1 cell differentiation from precursor from

precursor cells (Bray, 1985) and IFN- may be derived from NK cells (Scott,

42

1993), thereby may help in developing protective immunity in Leishmania

infection.

T cells

Activated T cells were demonstrated in the peripheral blood of patients with

Kala-azar (Raziuddin et al., 1992) secreting elevated levels of cytokines involved

in B cell growth and differentiation which may contribute to the polyclonal B cell

activation associated with Kala-azar.

CD8+ T cells

Cytotoxic T lymphocytes have traditionally been associated with resistance to

viral infections but three is now increasing evidence that these cells my also have

an important role in immunity to intracellular microbes (Kaufmann, 1988). This

may be due to direct cytotoxicity or through cytokine production. CD8+ T cells

produce IFN- and TNF- (Fong and Mosmann, 1990). Both these lymphokines

are known to be important in activating macrophages to kill Leishmania.

Therefore, CD4+T cells and CD8+T cells may interact to kill Leishmania-infected

macrophages both by production of lymphokines and by direct lysis. Therefore it

is likely that a greater role of CD8+T cells in the control of leishmaniasis will be

apparent.

CD4+T cells

Although a role for CD8+T cells in leishmaniasis appears to be likely the

important cell in mediating protective immunity in leishmaniasis is undoubtedly

the CD4+T cells. The subsets of CD4+T cell are directly related to both protective

immunity and disease exacerbation. Resistance of Leishmania infection and early

cure is associated with a T helper (Th)-1 cell response while disease progression

is related to Th2 type response.

43

For a clear understanding of the involvement of the subsets of CD4+T helper cells

in cell-mediated immune response in leishmaniasis a short overview of CD4+T

helper cells is given below.

CD4+T Helper Cells in Leishmaniasis

Evidences has accumulated that the differential immune response observed in

resistant and susceptible mice and healed and non-healed mice was due to

activation of different CD4+T cell subsets. Although there is a difference between

mice and humans in terms of the immune response to infection with different

Leishmania species, several important principles have emerged. Resolution of

leishmanial infection and protection against reinfection in susceptible humans and

mice is governed by the expansion of Leishmania-specific helper T cells of the

CD4+ Th1 cell type that produce IFN-. When present these cells activate

macrophages to kill intracellular amastigotes (Murray et al., 1983; Sundar et al.,

1994). IL-2 appears to play an important role in promoting the development of

protective Th1 responses (Murray and Hariprasad, 1995). TNF- also appears to

exert its leishmanicidal activity by activating macrophages, rather than acting

directly on the parasite (Liew et al., 1990). Membrane-associated TNF- on

Leishmania specific CD4+T cells may be important in sending activation signals

to infected macrophages, resulting in parasite killing (Sypek and Wyler, 1991).

During progressive systemic infections in mice, there is expansion of CD4+T cells

of the Th2 type that secrete IL-4, but not INF- or IL-2 inc response to

leishmanial antigens. IL-4 suppresses the development of murine Th1 response

and activation of macrophages by INF- (Liew et al., 1989). Similar reciprocal

activity of IL-4 and INF- was obtained with human monocytes infected with L.

donovani (Lehn et al., 1989). In human with Kala-azar, IL-10 rater than IL-4 may

be responsible for the suppression of potentially protective Th1 responses

44

(Holaday et al., 1993) but others suggested that IL-4 may play a more prominent

role than IL-10 in Indian kala-azar (Sundar et al., 1997).

2.9.6 Clinical features

Kala-azar has got various other names. These include Dum-dum fever, Sikari

disease, Burdwan fever and Shahib�s disease. However, the most commonly used

term is Kala-azar, which in Hindi means black sickness or black fever. The terms

originally referred to Indian. Kala-azar due to its characteristic symptoms,

blackening or darkening of the skin of the hands, feet face and the abdomen

(Lainson and Shaw, 1987). Typical Kala-azar usually occurs after an incubation

period of 2-6 months depending on the patient�s age, immune status and the

species of Leishmania. In endemic cases of Kala-azar, the disease is chronic and

onset is gradual. Although people of all ages are susceptible in the old world,

children below the age of 15 are more commonly affected with L. infantum being

largely responsible (Rab and Evans, 1995). The symptoms of Kala-azar vary

between individuals and according to geographical foci. Common symptoms

included high undulating fever often with 2-3 peaks in 24 hours and drenching

sweats which can easily be misdiagnosed as malaria. Chills, rigors, weight loss,

fatigue, cough, burning feet, insomnia, abdominal pain, joint pain, epistaxis and

diarrhoea might be associated. Most commonly observed clinical signs include

massive splenomegaly, hepatomegaly and anaemia. The duration of the disease

can be 1-20 weeks (Hashim et al., 1995). Splenic enlargement along with

hepatomegaly causes an abdominal protuberance in these patients.

While the disease progresses, the spleen extends well below the costal margin; it

is usually firm or hard in consistency but soft spleen may be encountered in acute

disease (Bryceson, 2000; Sundar et al., 2001). Late in the course, epistaxis and

gingival bleeding caused by severe thrombocytopenia may occur; oedema and

45

ascites may also develop. Jaundice with mildly elevated enzyme levels is rarely

seen and is considered to be a bad prognostic sign. Commonly encountered

laboratory findings include normocytic-normochromic anaemia, neutropenia,

thrombocytopenia, hypergammaglobulinemia as a result of polyclonal B cell

activation and hypoalbuminemia. Serum levels of hepatic transaminases may be

elevated (Totan et al., 2002; Haider et al., 2001; Peacock, 2001; Maltezou et al.,

2000; Minodier and Garnier, 2000).

2.10 Complications of Kala-azar

Kala-azar is commonly complicated by secondary infections, such as pneumonia,

bronchial infections, tuberculosis, malaria, diarrhoea or dysentery, viral

infections, bacterial skin infections, otitis media and Cancarum oris.

Thrombocytopenia may cause epistaxis or bleeding from other sites and this may

precede death. Leishmania enteritis may be a cause of diarrhoea and

malabsorption and pulmonary involvement may mimic pneumonia, Mortality is

related to immunosuppression causing secondary infections and hemorrhage and

in untreated cases mortality ranges from 75-95% (WHO, 1996; Peter, 2000).

2.11 Sequalae of Kala-azar and post Kala-azar dermal leishmaniasis (PKDL)

PKDL has been reported from India (Prasad, 1999) occur in approximately 10%

of cases after the treatment of Kala-azar. Lesions can develop at late as 1-2 years

after treatment for the original disease and manifest on the face, trunk or

extremities and may persist for as long as 20 years. In Africa, it has reported that

dermal lesions occur in only 2% of cases and tend to appear during or shortly after

the treatment and persist only for a few months (Zijlstra et al., 2000; Prasad,

1999). PKDL is characterized by a patchy and raised maculopapular rash and

changes in skin colour. Late manifestations are papules, papules or nodules. It

may be confused with lepromatous leprosy, fungal infections, diffuse cutaneous

46

leishmaniasis or other skin disorders. Pentavalent antimony at a dose of 20mg/kg/

day for 4 months or longer is used to treat Indian PKDL. In Ethiopia, Kenya and

Sudan, PKDL is treated for 2-3 months. Once lesions improve clinically treatment

may be stopped, as PKDL very rarely relapse (WHO, 1996).

2.12 Differential diagnosis of Leishmaniasis

The differential diagnosis of Kala-azar includes malaria, tropical splenomegaly

syndrome, schistosomiasis, cirrhosis with portal hypertension, African

trypanosomiasis, milliary tuberculosis, brucellosis, typhoid fever, bacterial

endocarditis, histoplasmosis, malnutrition, lymphoma and leukemia. Similarly

numerous primary and secondary skin disease and conditions are frequently

misdiagnosed as early lesions of cutaneous leishmaniasis. Some of the common

conditions that that should be differentiated from cutaneous leishmaniasis are

tropical ulcers due to other causes, impetigo, infected insect bits, leprosy, lupus

vulgaris, yaws, blastomycosis and skin cancer (Herwaldt, 1999). Mucocutaneous

leishmaniasis is sequelae of new world cutaneous leishmaniasis and results from

direct extension or hematogenous or lymphatic metastasis to the nasal or oral

mucosa. Paracoccidioidomycosis, polymorphic reticulosis, Wegener�s

granulomatosis, lymphoma, histoplasmosis, yaws, tuberculosis, nasopharyngeal

carcinoma and other destructive lesions are frequently misdiagnosed as early

lesions of mucocutaneous leishmaniasis (Herwaldt, 1999; El-Hassan and Zijlstra,

2001). Hence other diagnostic methods are required to confirm the clinical

suspicion (Herwaldt, 1999).

2.13 Laboratory diagnosis

Laboratory diagnosis of leishmaniasis can be made by the following:

(i) demonstration of parasite in tissues of relevance by light microscopic

examination of the stained specimen, in vitro culture, or animal inoculation; (ii)

47

detection of parasite DNA in tissue samples; or (iii) immunodiagnosis by

detection of parasite antigen in tissue, blood, or urine samples, by detection of

nonspecific or specific antileishmanial antibodies (immunoglobulin), or by assay

for Leishmania-specific cell-mediated immunity (Sundar and Rai, 2002).

2.13.1 Microscopic examinations

The commonly used method for diagnosing Kala-azar has been the demonstration

of parasites in splenic or bone marrow aspirate. The presence of the parasite in

lymph nodes, liver biopsy, or aspirate specimens or the buffy coat of peripheral

blood can also be demonstrated. Amastigotes appear as round or oval bodies

measuring 2 to 3 µm in length and are found intracellularly in monocytes and

macrophages. In preparations stained with Leishman�s stain, the cytoplasm

appears pale blue, with a relatively large nucleus that stains red. In the same plane

as the nucleus, but at a right angle to it, is a deep red or violet rod-like body called

a kinetoplast.

After identification, parasite density can be scored microscopically by means of a

logarithmic scale ranging from 0 (no parasite per 1,000 oil immersion fields) to +6

(>100 parasites per field) (Chulay and Bryceson, 1983). The sensitivity of the

bone marrow smear is about 70% or lower (Boelaert et al., 1999 and Chowdhury

et al., 1993). Splenic aspirate, though associated with rise of fatal hemorrhage in

inexperienced hands, is one of the most valuable methods for diagnosis of kala-

azar, with a sensitivity exceeding 90% (Zijlstra et al., 1992 and Kager et al.,

1983). It required no special equipment, from the patient�s standpoint is generally

preferable than the more painful bone marrow aspirate, and has proven to be safe

and relatively easy to perform in experimented hands. For patients suspected of

have Kala-azar, splenic aspirate can be performed even when spleen is not

palpable, after demarcating the area of splenic dullness by percussion. The only

48

risk of splenic puncture is bleeding from a soft and enlarged spleen. Occasionally

amastigotes have also been demonstrated in liver biopsy (50-80% sensitivity) and

lymph node aspirate (56% sensitivity) (Zijlstra et al., 1992).

Blood buffy coat:

Another interesting field for definitive diagnosis of Kala-azar might be the

detection of LD body from buffy coat smear of peripheral blood. Because, when

blood is settled in presence of an anticoagulant, the WBCs are deposited and

concentrated in buffy coat at the interface of RBCs and plasma (Shamszzaman et

al., 2007). Under all aseptic precautions (cleaning the area with povidone iodine

followed by adequate rubbing by alcohol pad containing 60% isopropyl) 3 ml of

venous blood will be collected from median ante-cubital or appropriate veins by

gentle suction. The collected blood sample will immediately be introduced into a

3.2% Tri-sodium citrate coated vacutainer. In a sterile round bottom disposable

test tube (10 ml capacity), 2 ml of Histopaque will be pipette carefully. Then 2 ml

of citrated blood from the vacationer will be poured slowly by the side of the test

tube so that the blood completely overlay the separation fluid with out any

mixing. The test tube will now be centrifuged for 15 minutes. 3000 rpm referable

in a swinging centrifuge machine. After centrifugation, a thick buffy coat will be

seen in the interface of plasma and Histopaque. RBCs will be settled at the bottom

part of the test tube. Now the plasma will be gently sucked by a micropipette of

1ml capacity and will be preserved in an eppendrof tube. Then by another of 100-

200 µl capacity, the buffy coat material will be sucked out very gently. Part of the

material will be used in another eppendrof containing 0.5 ml of 95% alcohol for

analysis by PCR (DGHS, 2009).

If buffy coat is considered for making a smear, it is supposed to show high content

of WBCs including monocytes. The monocytes parasitized by LD bodies, if

49

present, will be higher in the smear. Thus the rate of LD body positivity is

expected to be higher (Shamszzaman et al., 2007).

2.13.2 Culture of Leishmanial parasites

Culture of parasite and improve the sensitivity of parasite isolation but

Leishmania culture is rarely needed in routine clinical practice. However, cultures

are required for (i) obtaining a sufficient number of organisms to use an antigen

for immunologic diagnosis and speciation (ii) obtaining parasites to be used in

inoculating susceptible experimental animals (iii) in vitro screening of drugs (iv)

accurate diagnosis of the infection with the organism. Leishmania strains can be

maintained as promastigotes in artificial culture medium. The culture media used

may be monophonic (Schneider�s insect medium, M199 or Grace�s medium) or

diphasic (NNN and Tobies medium). Most preferable diphasic medium is that

which contain modified diphasic rabbit blood agar overlaid with RPMI 1640 from

Gibco BRI, Grand Island, N.Y for primary isolation and M199 medium

containing 20% fetal calf serum to amplify parasite numbers (Sundar et al., 2001).

Hockmeyer�s medium, which is Schneider�s commercially prepared culture

medium supplemented with 30% heat-inactivated fetal calf serum with 100 IU of

penicillin and 100 µg of streptomycin, is simple to use and satisfactory for

diagnosis of Kala-azar but it is expensive. Culture tubes are inoculated with 1 to 2

drops of bone marrow or splenic aspirate and incubated at a temperature between

22 0C and 280C. The tubes are examined weekly for the presence of promastigotes

by phase contrast microscopy or by wet mount of culture fluid for 4 weeks before

being discarded as negative. If promastigotes are present, they are maintained by

weekly passage to fresh medium. Blood can also be used to isolate the parasite but

the method is slow and takes longer. Aseptically collected blood (1 to 2 ml) is

diluted with 10 ml of citrated saline and the cellular deposit obtained after

centrifugation is inoculated in culture media. Contamination of the culture media

50

by bacteria of yeast species or other fungi usually complicates the culture but can

be avoided by use of good sterile techniques and be the addition of penicillin (200

IU/ml), streptomycin (200µg/ml) and 5-flucytosine (500µg/ml) to the medium

(Schur and Jacobson, 2001). In vitro culture of the amastigotes is done for

chemotherapeutic studies and to study the interrelationship of the amastigotes and

macrophages. The amastigotes are grows in tissue or macrophage culture. These

cell lines are produced from (i) human peripheral blood monocytes by density

sedimentation with lymphocyte separation medium from LSM; Organon-Teknika,

Durham, N.C (Grogl et al., 1993) (ii) macrophage cell lines, e.g., P388D and

J774G8 lines from mice; and (iii) dog sarcoma and hamster peritoneal exudates of

cell line, in which case continuous culture can be achieved (Schur and Jacobson,

2001).

Species recognition:

In areas of endemicity, recognition of species of Leishmania is rarely required.

However, identification of an organism to the species level is helpful

epidemiologically and is also important for the treatment and prognosis

determination for global travelers who are not immune to the parasite and tend to

develop unusual manifestations of the disease (Magill et al., 1993). Identification

of species of the L. donovani complex is particularly difficult, because

morphologically the species are almost indistinguishable from each other. For

species level identification, a large amount of promastigotes is obtained by culture

of the organism and the species-specific isoenzyme pattern is analyzed by

cellulose acetate electrophoresis (Kerutzer et al., 1993). Typing of washed live

promastigotes by direct agglutination test with species-specific monoclonal

antibodies is another highly sensitive taxonomic tool frequently utilized for this

purpose (Jaffe and Sarfstein, 1987). Species-level identification can also be done

by analysis of amplified minicircle kinetoplast DNA (kDNA), by choosing

51

primers from conserved regions of different Leishmania species kDNA

minicircles (Sacks et al., 1995; Smyth et al., 1992). Yet another method used for

identification of species of Leishmania is the analysis of the in vitro promastigotes

released antigenic factors, which are different for different leishmanial species

(IIg and Stierhof, 1994).

2.13.3 Immune diagnosis

Specific serological tests

Antigen Detection

The antigen detection is an ideal method of diagnosing an infection. Antigen

levels are expected to broadly correlate with the parasite load as well. Antigen

detection is more specific than antibody-based immunodiagnostic tests (De

Colmenares et al., 1995; Vinayak et al., 1994). This method is also useful in the

diagnosis of disease in cases where there is deficient antibody production (as in

AIDS patients). De Colmenares et al., (1995) from Spain have reported two

polypeptide fractions of 72-75kDa and 123 kDa in the urine of kala-azar patients.

The sensitivities of the 72-75-kDa fractions were 96% and the specificities were

100%. Besides, these antigens were not detectable within 3 weeks of anti-kala-

azar treatment, suggesting that the test has a very good prognostic value.

However, detection of antigen in the patient�s serum is complicated by the

presence of high level of antibodies, circulating immune complexes, serum

amyloid, rheumatoid factor and autoantibodies; all of which may mask

immunologically important antigenic determinants or competitively inhibit the

binding of free antigen (Senaldi et al., 2001). A new latex agglutination test

(KATEX) for detecting leishmanial antigen in urine of patients with Kala-azar has

showed sensitivities between 68 and 100% and a specificity of 100% in

preliminary trials. The antigen is detected quite early during the infection and the

results of animal experiments suggest that the amount of detectable antigen tends

52

to decline rapidly following chemotherapy. The test performed better than any of

the serological tests when compared to microscopy. Large field trials are under

way to evaluate its utility for the diagnosis and prognosis of Kala-azar (Attar et

al., 2001). Whether the test the applications for the detection of asymptomatic

cases of Kala-azar and monitoring therapy is yet to be confirmed. There are no

antigen detection systems currently available for CL and MCL.

Enzyme Linked Immunosorbent Assay:

The Enzyme Linked Immunosorbent Assay (ELISA) is a valuable tool in the

serodiagnosis of leishmaniasis. The test is useful for laboratory analysis as well as

for field applications. However, the sensitivity and specificity of ELISA is greatly

influenced by the antigen used. More recently, several recombinant antigens like

rGBP form L. donovani, rORFF from L. infantum, rgp63, rK26 and rK39 from L.

chagasi have been developed and tested (Montoya et al., 1997; Martin et al.,

1998; Jensen et al., 1999; Rajasekariah et al., 2001; Raj et al., 1999; Bhatia et al.,

1999). Of these, the rK39 antigen is found to be highly sensitive and predictive of

onset of disease manifestation of Kala-azar patients. In contrast, it doe not show

detectable antibodies in CL or MCL. The antibody titres to this antigen directly

correlate with active disease and have potential in monitoring the chemotherapy

and in predicting the clinical relapse. In addition rK39 ELISA has a high

predictive value for detecting Kala-azar in immunocompromised person, like

those with AIDS. A kit (InBios, USA) using this antigen and based on lateral flow

is now commercially available in the form of antigen-impregnated nitrocellulose

paper strips adapted for the use under field conditions. The rK39 strip test has

been found highly sensitive has a reliable indicator of KA in Indian subcontinent

(Burns et al., 1993; Singh et al., 1995; Singh et al., 2002; Houghton et al., 1998).

Due to lack of facilities and expertise to perform skin biopsy, smears and cultures

the proper diagnosis of cutaneous leishmaniasis remains under reported. The use

53

of serological tools in the diagnosis of CL has also been assessed by a number of

studies. ELISA using few recombinant antigens viz., gene B protein (GBP) from

L. major recombinant major surface glycoprotein, gp63, from L. major and 2

recombinant proteins, T26-U2 and T26-U4 from Viannia peruviana have been

tried but have been found less suitable for diagnosing CL (Schalling et al., 2001;

Zijlstra et al., 2001). Laboratory diagnosis of MCL has also taken a back seat and

most of the cases are diagnosed clinically.

Antibody detection

Conventional methods for antibody detection included gel diffusion, complement

fixation test, indirect haemagglutination test, IFA test, and countercurrent

immuoelectrophoresis (Bray et al., 1985, Haldar et al., 1981 and Hockmeyer et

al., 1984).

Indirect fluorescent antibody test (IFAT):

The Indirect fluorescent test is one of the most sensitive tests available. The test is

based on detecting antibodies which are demonstrated in the very early stages of

infection and are undetectable six to nine months after cure. If the antibodies

persist in low titers, it is a good indication of a probable relapse a probable

relapse. Titers above 1:20 are significant and above 1: 128 are diagnostic

(Davidson, 1998). There is a possibility of a cross reaction with trypanosomal

sera. However this can be overcome by using Leishmania amastigotes as the

antigen instead of the promastigotes (Weigle et al., 1987). Although this test is

more sensitive (96%) and specific (98%) than soluble antigen of ELISA, it is

cumbersome and not suitable for field conditions (Sassi et al., 1999).

Direct agglutination test (DAT):

The direct agglutination test is a highly specific and sensitive test. It is

inexpensive and simple to perform making it ideal for both field and laboratory

54

use. The method uses whole, stained promastigotes either as a suspension or in a

freeze-dried form. The freeze-dried form is heat stable and facilitates the use of

DAT in the field. However, the major disadvantage of DAT is the relative long

incubation time of 18 hours and the need for serial dilutions of serum (Gari-

Toussaint et al, 1994; Schallig et al., 2001). DAT has no prognostic value too.

The test may remain positive for several years after cure. Recently, Schoone et al.

(2001) have developed a fast agglutination-screening test (FAST) for the rapid

detection (<3 hours) of anti-leishmania antibodies in serum samples and on blood

collected on filter paper. The FAST utilizes only one serum dilution leading to

qualitative results. It offers the advantages of DAT based on the freeze-dried

antigen regarding reproducibility, specificity and sensitivity.

DAT has been evaluated for the diagnosis of CL using L. major promastigotes and

was shown to be highly (90.5%) specific and sensitive (Schallig et al., 2001).

Immuno-chromatography test (ICT):

A recombinant antigen, rK39 has been shown to be specific for antibodies in

patients with Kala-azar caused by members of the L. donovani complex (Badaro

et al., 1996; Bern et al., 2000; Burns et al., 1993). This antigen, which is

conserved in the kinesin region, is highly sensitive and predictive of the onset of

acute disease. The antigen is derived from L. chagasi, which in the Untied States

is used for veterinary purposes, thought it is not approved for human use. High

antibody titers in immunocompetent patients with Kala-azar have been

demonstrated. This antigen has been reported to be 100% sensitive and 100%

specific in the diagnosis of Kala-azar and PKDL by ELISA (Kumar et al., 2001;

Singh et al., 1995). Another important facet of anti-rK39 antibody is that the titer

correlates directly with the disease activity, indicating its potentiality for use in

predicting response to chemotherapy. It was previously shown that anti-rK39

55

antibody titers were 59-fold higher than those of antibody against CSA at the time

of diagnosis, and with successful therapy, it fell sharply at the end of treatment

and fell further during follow-up monitoring. In patients who experience disease

relapse, the titer rose steeply again (Kumar et al., 2001). The diagnostic and

prognostic utility of rK39 for HIV-infected patients has also been demonstrated

(Houghton et al., 1998). Because of the conditions prevailing in areas of

endemicity, any sophisticated method cannot be employed on a wider scale. There

is a need for a simple rapid and accurate test with good sensitivity and specificity,

which can be used without any specific expertise. A promising ready to use

immunochromatographic strip test based on rK39 antigen has been developed as a

rapid test for use in difficult field conditions. The recombinant antigen is

immobilized on a small rectangular piece of nitrocellulose membrane in a band

form and goat-anti-protein A is attached to the membrane above the antigen band.

After the finger is pricked, half a drop of blood is smeared at the tip of the strip,

and the lower and end of the strip is allowed to soak in 4 to 5 drops of phosphate-

buffered saline, placed on a clean glass slide or tube. If the antibody is present, it

will react with the conjugate (protein A colloidal gold) that is predried on the

assay strip. The mixture moves along the strip by capillary action and reacts with

rK39 antigen on the strip, yielding a pink band. In the strip of patients who are

infected, two pinkish lines, appear in the middle of the nitrocellulose membrane

(the upper pinkish band serves as a procedural control). In the first extensive field

trial in 323 patients, we found the strip test to be 100% sensitive and 98% specific

(Sundar et al., 1998). Several studies from the Indian subcontinent reported the

test to be 100% sensitive (Bern et al., 2000; Sundar and Rai, 2002; Sundar et al.,

1998). However, when evaluated in Sudan, the sensitivity of the test was only

67%. In the Sudan study, all the parasitologically confirmed Kala-azar patients

who tested negative by the rK39 strip test showed IgG against rK39 by micro-

56

ELISA at lower titers (Zijlstra et al., 2001). In a study done in southern Europe,

the rK39 strip test results were positive in only 71.4% of the cases of Kala-azar

(Jelinek et al., 1999). These differences in sensitivity may be due to differences in

the antibody responses observed in different ethnic groups (Singh et al., 1995).

When tested for PKDL, the test had 91% sensitivity (Salotra et al, 2001). High

levels of specificity (97 to 100%) have been reported uniformly for this test;

however, with a later version of the rK39-treated strips, some (12.5%) healthy

endemic control subjects also tested positive ( Sundar and Rai, 2002). While such

reactions might be considered to be false positive, these probably represent

subclinical infections: PCR assay for L. donovani was positive in a few of these

cases (Salotra et al., 2001; Sundar and Rai, 2002). Anti-rK39 IgG may be present

in serum for an extended period after successful treatment for Kala-azar; thus,

patients with suspected relapse of Kala-azar with a past history of infection would

not be candidates for diagnosis by strip testing. Another drawback of this format

is that an individual with a positive rK39 strip test result may suffer from an

illness (malaria, typhoid fever or tuberculosis) with clinical features similar to

those of Kala-azar yet be misdiagnosed as suffering from Kala-azar. Besides these

limitations, the rK39 ICT strip test has proved to be versatile in predicting acute

infection. It is the only available format for diagnosing Kala-azar with acceptable

sensitivity and specificity. It is also inexpensive and simple (Sundar and Rai,

2002).

Complement Fixation Test (CFT):

Complement fixation test was introduced more than 60 years ago in the diagnosis

of Kala-azar (Smith et al., 1984). Since then it has been used as a serodiagnostic

tool in different countries. Although, CFT becomes positive in the early stage of

infection (in 3 weeks) it is a non-specific test. Sensitivity of this test varies with

the source and method of extraction of antigen and also with different batches of

57

antigen (Aikat et al., 1979) and false positive results are encountered. In addition,

the test has shown to be of limited value in routine laboratory diagnosis (Duxbury

and Sadun, 1964) and large-scale epidemiological studies (Hommel et al., 1978).

Due to its complicated procedure that test can only be performed in reference

laboratories (Rahman and Islam, 1979).

Immunoblotting:

Most of the work concerning the use of immunoblotting in the diagnosis of

leishmaniasis has been done on Kala-azar. In CL, anti-leishmanial antibodies

though detectable, are present in low titres, Hence Immunoblotting is not widely

adopted for diagnosing CL. The biggest advantage of immunoblotting, as a

general rule, is that various antigens expressed and antibodies recognized during

the course of infection can be documented. It also has an advantage of permanent

documentation. However, the technique is not user-friendly and limited only to

research laboratories (Herwaldt, 1999; Weigle et al., 1987; Maalej et al., 2003;

Brito et al., 2000).

Non-specific tests

Formol gel test

The formol get test has the advantage of being cheap and simple to perform.

Serum obtained from about 5 ml of blood is mixed with one drop of 30%

formaldehyde. A positive reaction is shown if the mixture solidifies and forms a

white opaque precipitate within 20 minutes. A positive test can not be detected

until 3 months after infection and becomes negative 6 months after cure. The test

is non specific since it is based on detecting raised levels of IgG and IgM which

also result from African trypanosomiasis, malaria and schistosomiasis (WHO,

1991).

58

Skin testing (Cell-mediated immunity)

Leishmanin Skin Test (LST):

Delayed hypersensitivity is an important feature of cutaneous forms of human

leishmaniasis and can be measured by the leishmanin test, also known as the

Montenegro reaction. Leishmanin is a killed suspension of whole (0.5-1×10/ml)

or disrupted (250 g protein/ml) promastigotes in pyrogen-free phenol saline

(Palma and Gutierrez, 1991). No cross-reactions occur with Chagas� disease but

some cross-reactions are found with cases of glandular tuberculosis and

lepromatous leprosy. Leishmanin skin test in usually used as an indicator of the

prevalence of cutaneous and mucocutaneous Leishmaniasis in human and animal

populations and successful cures of Kala-azar (Weigle et al., 1987; Singh, 1999;

Manson-Bahr, 1987). During active KA disease there will be no or negligible cell

mediated immune response. However, the leishmanin antigen is not commercially

available and no field study has been carried out in India.

2.13.4 Molecular method

Microscopy and culture have the limitations of low sensitivity and are time

consuming. The immunological methods fail to distinguish between past and

present infections and are not very reliable in immunocompromised patients.

While the molecular approach is capable of detecting nucleic acids unique of the

parasite, it would address these limitations. A variety of nucleic and detection

methods targeting both DNA and RNA have been developed. Recently molecular

probes, using kinetoplast DNA (kDNA), ribosomal RNA (rRNA), mini exon

derived RNA (med RNA) and genomic repeats have been evaluated and used to a

much higher sensitivity and specificity. The distinct advantages of DNA

hybridization are that large numbers of samples can analyzed quickly without

compromising efficiency and many different types of samples can be processed

59

including blood spots, tissue samples, splenic and bone marrow aspirates (Wilson,

1995). The probes are more specific since they are made to target regions on the

kDNA or rRNA, which are unique to a particular Leishmania species, complex or

isolate (Williams et al., 1995). A probe has been developed to recognize the

genomic DNA repeat Lmer2 that is specific for L. donovani. This probe has been

used to diagnose infection in human Kala-azar patients and in P. martini, the

sandfly vector of L. donovani (Wilson, 1995). The early DNA probes were

labeled with radioactive 32p, which were easy to use and gave high signals with

low background interference. However, these were for laboratory use only and

were not suitable as diagnostic kits, since 32p has a short half-life of about 14 days

and being radioactive they presented safety problems. Recently, a

chemiluminescent hapten digoxigenin labeled kDNA probes have been

developed. These probes are as sensitive as the 32p labeled probes (can detect as

low as 100 parasites) and are stable enough to use in diagnostic kits, which are

more practical in the field. However, high background signals make result

interpretation difficult (Wilson, 1995; Williams et al., 1995). The latest

developments in molecular diagnostic technology have come about as a direct

result of the advent of the polymerase chain reaction (PCR). The PCR is able to

amplify small amounts of DNA or RNA to larger usable quantities (Nuzum et al.,

1995; Wilson, 1995). Although the PCR is able to detect a single copy of target

DNA, repeat sequences are used to improve sensitivity. The PCR assay can detect

parasite DNA or RNA a few weeks ahead of appearance of any clinical signs or

symptoms. Different DNA sequence in the genome of Leishmania like it�s region,

gp63 locus, telomeric sequences, sequence targets in rRNA genes such as 18s

rRNA and SSU-rRNA and both conserved and variable regions in kinetoplast

DNA (kDNA) minicircles are being used by various workers (Santos-Gomes et

al., 2000; El Tai et al., 2001; Pizzuto et al., 2001; Wortman et al., 2001; Ostria

60

and Tezeda, 2002). An improved PCR solution hybridization enzyme linked assay

(PCR-SHELA) was developed and has been used to diagnose infection of L.

donovani in patients in India, Kenya and Brazil with 90% sensitivity and 100%

specificity (Nuzum et al., 1995). The PCR-SSCP technique has been developed

for the detection of sequence variation in rRNA genes within the L. donovani

species. In addition, it can be performed easily and rapidly from clinical samples

without prior need of cultivation of the parasite. PCR assay has also been used for

post-therapeutic follow-up and for detection of relapses among HIV-infected

patients using SSU rRNA gene target. The use of fluorogenic real-time PCR using

SSU-rRNA gene added with complete automation has made quantification of

parasite burden possible. Beside it is a rapid, sensitive and highly specific test

(Attar et al., 2001; Santos Gomes et al., 2000; El Tai et al., 2001; Pizzuto et al.,

2001; Wortman et al., 2001). Recently, primers was developed that can

differentiate the Indian strains causing Kala-azar and PKDL. An Alu-PCR-like

amplification was performed from the cultured L. donovani isolated from Kala-

azar and PKDL patients. The banding pattern of PCR amplicons could clearly

group all the PKDL strains in one group while Kala-azar stains had intro-species

heterogeneity as reported by us earlier (Ostria and Tezeda, 2002).

2.14 Treatment of Kala-azar

The main objectives in the treatment of kala-azar are as follows:

To cure the patient of an intracellular parasitic infection

To prevent relapse and the development of unresponsiveness

To keep hospitalization and treatment cost to minimum

The first-line drugs:

After initial assessment of the patient, start with standard first line drugs

pentavalent antimonials, sodium stibogluconate (SSG). The aim of the first line

61

therapy is to supply an adequate daily dose of SSG for long enough to achieve

parasitological cure without relapse (Chowdhury et al., 1993).

The drugs are administered parentally and are the safest currently available drugs

since they are rapidly excreted by the kidneys and there is virtually on

accumulation in the body. Potential side effects include nausea, vomiting,

diarrhoea, ECG changes and convulsions. Sodium stibogluconate is usually

administered at a dose of 20 mg/Kg body weight daily in a single dose for 28

days. Relapse cases can be treated with same therapy for 40-60 days (Abdullah,

2002).

The second-line drugs:

Second line drugs are used for resistant Kala-azar cases. Drug resistance can be

primary or secondary. Resistance can be caused by delayed diagnosis (prolong

duration of illness), interrupted and low dose treatment, immunological failure,

emergence of resistant strains of parasites and leishmaniasis associated with

AIDS. Leishmaniasis in AIDS, mainly reported from southern Europe, now is

observed in other part of the world, including South America, Asia and North

Africa. Patients with an HIV co-infection can develop unusual manifestations of

leishmaniasis. Parasitologic diagnosis by peripheral blood smear and buffy coat

smear is more rewarding in HIV co-infection because parasites are found more

commonly in the monocytes of these patients. Resistance to stibogluconate should

be treated with alternate agents, such as:

Liposomal amphotericin (0.5-3 mg/kg) on alternate days until a dose of 20

mg/kg. Liposomal amphotericin is a lipid complex with amphotericin that

is more active than amphotericin B. The 3 preparations formulated are

amphotericin B lipid complex, liposomal amphotericin B and amphotericin

B colloidal dispersion. Cure rates of more than 90% have been observed in

62

various studies. The high cost of these drugs is a disadvantage to its use in

areas where Kala-azar is prevalent.

Pentamidine (2-4 mg/kg) on alternate days for 15 doses. Pentamidine is

available in 2 preparations, pentamidine isethionate (Pentam 300) and

pentamidine dimethane sulphonate (Lomidine). Lomidine given in the

same schedule is more effective than pentamidine isethionate. The serious

adverse effects associated with pentamidine include fall in blood pressure

with an intravenous injection, hypoglycaemia, pancreatitis and reversible

renal dysfunction.

Combination of stibogluconate with drugs such as aminosidine and

interferon gamma, also has produced good results in cases showing poor

response to stibogluconate therapy alone. Aminosidine, an

aminoglycosides identical to paromomycin also has been found effective in

trials from India (http://www.emedicine.com).

Oral drugs have obvious advantages over injectables, and several oral

formulations lie allopurinal, fluconazole and autovaquone have been tested.

Stimaquine and miltefosine are two promising oral anti-leishmanial compounds.

But recent from Brazil showed poor efficacy of stimaquine compounded with

nephrotoxicity. Miltefosine, an alkyl phospholipids, developed as an antitumour

agent, is the first oral compound which has been successfully developed in India

for the treatment of Kala-azar. A daily dose of 100-150 mg for four weeks cured

>95% patients. Recommended daily dose is 100 mg for adults weighing more

than 25 kg and 50 mg for those weighing <25 kg for four weeks (Sundar, 2003).

Kala-azar treatment failure (KATF): When kala-azar does not respond to usual

therapy with sodium stibogluconate. It is usually due to inappropriate or sub-

63

optimal dose or irregular therapy. Treatment is usually with second line drugs like

resistant kala-azar cases.

Pentavalent antimonials remain the drugs of choice for treating PKDL. Injection

sodium stibogluconate at a dose of 20 mg/kg of body weight daily for 20 days.

Then 10 days interval. This is one cycle. Total 6 cycles are needed. If a second

course is required, it should be given after an interval to two months. Pentamidine

is infective in PKDL (Abdullah, 2002).

Diet: High protein and high calorie diet during the course of treatment is required

(http://www.emedicine.com).

The emergence of drug resistance in Leishmania parasites is a major obstacle to

their control. The lack of response to pentavalent antimonials in Kala-azar has

been a problem for many years and is increasing world wide, occurring in about

5-70% patients in some endemic areas (Quellette and Papadopoulou, 1993).

Prognosis to kala-azar depends upon the immune status of the host and effective

treatment. In a person who is non-immune to kala-azar, the introduced

promastigote changes to amastigote, increase in number and spread to distant

organs without hindrance and manifests the disease. If such as patient is left

untreated their mortality is very high from 90 to 95 percent in adults and from 75

to 80% in children. Death usually follows within 2 years of the onset mainly due

to some complications such as diarrhoea, amoebic and bacillary dysentery,

pneumonia, pulmonary percentage of cases, kala-azar may be self-limiting and

spontaneous cure is possible (Chowdhury et al., 1993).

2.15 Control measures for Kala-azar

Elimination of sandfly vectors:

In endemic areas such as Bangladesh and India sandfly control is often combined

with malaria control and Brazil with malaria and chagas disease control

64

programmes (WHO, 1997; Al-Masum et al., 1995). This is cost effective method

given the expense of mass spraying with chemicals such as DDT, Malathion,

Fenitrothion Propoxur and Diazinon. In endemic areas this has to be an on-going

process and continued surveillance is required to keep the vector populations low

(Al-Masum et al., 1995). In epidemics large scale spraying is required. Spraying

should particularly include animal shelters, inside of buildings and the immediate

surrounding areas. Self-protection is important, several methods are available to

aviod being bitten by sandflies including repellents such as diethyltoluamide

(DEET) applied to the exposed areas of the body and clothing. Fine mesh screens

(less than 16-mesh) can be applied to doors and windows and bed nets should be

used impregnated with insecticides such as permethrin and deltamethrin.

Mosquito nets are not effective since sandflies can get through the holes.

Elimination of reservoir hosts:

In areas of anthroponotic foci of Kala-azar active case detection and treatment is

imperative, especially asymptomatic seropositive patients. A recent study in Israel

showed that the asymptomatic or sub-clinical cases are 4-30 times greater than

those with the Kala-azar symptoms (Ephros et al., 1994). The IFAT, ELISA,

DAT, the western blot and the Leishmania skin test have all been useful in

detecting seropositive cases of Kala-azar (Rab and Evans, 1995; Shiddo et al.,

1995). In endemic areas of zoonotic Kala-azar, elimination of wild animals and

stray doges can be carried out by shooting and by using poisoned baits

impregnated with strychnine. Screening and treatment of domestic animals

especially dogs is recommended using the various available techniques including

the IFAT, DAT and ELISA (Shiddo et al., 1995).

65

CHAPTER 3

MATERIALS AND METHODS

3.1 Place and period

The study was conducted in the department of Microbiology, Mymensingh

Medical College, during the period from July 2009 to June 2010. Cases were

selected from Mymensingh Medical College Hospital and Trishal Thana Health

Complex.

3.2 Type of the study

The study was designed as a cross sectional study.

3.3 Study population

106 cases and 44 healthy controls were included in this study.

3.4 Subjects

Group I (confirmed Kala-azar cases): Clinically suspected febrile patients,

subsequently confirmed as Kala-azar by detection of LD bodies from bone

marrow aspirates by microscopy (n=41).

Group II (Serologically diagnosed Kala-azar cases): Clinically suspected

febrile patients, subsequently diagnosed as Kala-azar by detection of

antileishmanial antibodies using rK-39 antigen based ICT (n=65).

Group III (Controls): People from endemic area those were free from fever,

physically healthy and comparable to age and sex of the Kala-azar cases were

included as healthy controls (n=44).

66

3.4.1 Inclusion criteria

A total of 106 patients (clinical isolates) irrespective of age and sex were selected

from Mymensingh Medical College Hospital and Trishal Thana Health Complex

on the basis of following criteria:

Patients had history of prolonged low grade irregular fever.

Weight loss

Normal or increased appetite

Pallor

Bleeding

Organomegaly

Blackening of skin

3.4.2 Exclusion criteria of cases

Febrile persons showing negative results either in ICT or LD body were excluded

from the study.

3.5 Controls

People from endemic area those were free from fever, physically healthy and

comparable to age and sex of the Kala-azar cases were included as healthy

controls.

3.6 Data collection and analysis

Relevant history, clinical findings, laboratory records and follow-up of every case

was collected and recorded in a per-designed data sheet. Subsequently, data were

analyzed by computer programmed SPSS version 12.0.

3.7 Collection of specimen

Sample or specimen: Blood, bone marrow.

67

(a) Collection of bone marrow aspirates: The preferred site for bone marrow

aspiration include sternum preferably manubrium of the sternum, iliac crest and

sometimes tibia. The patient was admitted in medicine and pediatric unit in

Mymensingh Medical College Hospital. The purpose of bone marrow collection

was explained clearly to the patient and the attendant. After adequate motivation,

written consent was taken from the legal guardian or from the patient. Keeping the

patient lying on the side, site was palpated and foxed. The area was cleaned by

tincture iodine and rectified spirit. Injection Jasociane (1%) 1.5-2.0 ml was pushed

into the site as local anesthetic. Allowing 5-10 minutes time, appropriate bone

marrow needle was inserted up to the marrow cavity. Then up to 1 ml of marrow

material was aspirated by gentle suction through a 10 ml disposable syringe. After

completing aspiration, the syringe was removed from the needle and the needle

was drawn out. The local wound was sealed by sterile gauge and microspore

adhesive tape. Immediately after aspiration of bone marrow, thin film of the

aspirate was made on at least three microscopic slides. Films were allowed to dry

in air and the back of the slide was rubbed rapidly with a finger to slightly warm

the glass and drive off moisture (Dey et al., 1998). With proper labeling bone

marrow smear was carried to the laboratory for staining and microscopic

examination.

(b) Collection of blood: Under all aseptic precautions (cleaning the area with

povidone iodine followed by adequate rubbing by alcohol pad containing 60%

isopropyl) 5 ml of venous blood was collected from median ante-cubital or

appropriate veins by gentle suction. The collected blood sample was immediately

be introduced into a 3.2% Tri-sodium citrate coated vacutainer. Rest of the blood

was used to prepare peripheral blood film, ICT strip test, estimation of

hemoglobin and ESR. Immediately after collection with proper labeling, blood

was carried to the laboratory for buffy coat preparation.

68

3.8 Laboratory methods

3.8.1 Staining of marrow film: At first marrow film was covered with Leishman

stain (Appendix II) and kept for one minute. Then double quantity of distilled

water was added and kept for 12 minutes. Finally more distilled water was flowed

over the marrow film for washing and dried in the air (Duguid, 1996).

3.8.2 Microscopic examination of marrow film: At least 1000 fields per slide

preferably around the edges of the preparation were examined under oil

immersion lens to detect amastigote from of Leishmania donovani. Leishmania

amastigote appeared as small round or oval organisms about 3µm x 5 µm size

inside or outside macrophages. Each amastigote was containing a red-mauve

nucleus, a smaller more deeply staining red-mauve kinetoplast and pale blue

cytoplasm. The particular diagnostic feature to look for in LD bodies was the

presence of nucleus and kinetoplast. After identification, parasite density can be

scored microscopically by means of a logarithmic scale ranging from 0 (no

parasite per 1,000 oil immersion fields) to +6 (>100 parasites per field). The

grading of parasitemia is done by the criteria suggested by the WHO (10x

eyepiece and 100x oil immersion lens):

6+ >100 parasites/fields

5+ 10-100 parasites fields

4+ 1-10 parasites fields

3+ 1-10 parasites/ 10 fields

2+ 1-10 parasites/ 100 fields

1+ 1-10 parasites 1000 fields

0 0 parasites 1000 fields (Source : WHO TRS 793)

3.9 Making buffy coat (density gradient centrifugation)

In a sterile round bottom disposable test tube (10 ml capacity), 2 ml of Histopaque

(Appendix II) pipetted carefully. Then 2 ml of citrated blood from the vacutainer

69

was poured slowly by the side of the test tube so that the blood completely

overlay the separation fluid without any mixing. The test tube was centrifuged for

15 minutes. 3000 rpm referable in a swinging centrifuge machine.

3.9.1 Taking buffy coat material and preparation of smears

After centrifugation, a thick buffy coat was seen in the interface of plasma and

Histopaque. RBCs were settled at the bottom part of the test tube. Now the plasma

was gently sucked by a micropipette of 1ml capacity and was preserved in an

eppendrof tube. Then by another of 100-200 µl capacity/micropipette, the buffy

coat material was sucked out very gently. Part of the material was used in another

eppendrof containing 0.5 ml of 95% alcohol for analysis by PCR (DGHS, 2009).

3.9.2 Staining of buffy coat smears

Buffy coat films were stained by Leishman stain.

Procedure of Leishman staining

A air dried thin film was made and placed on a staining rack and flooded with

Leishman stain. The slide was left for 2 minutes to fix. Double amount distilled

water was added over the flooded stain from a plastic wash bottle for better

mixing of the solution. The stain was left for 10 minutes. The stain was washed

with running tap water and air dried again the smear to examine under oil

immersion objectives (DGHS, 2009).

3.9.3 Microscopic examination of the buffy coat film

At least 1000 fields per slide were examined under oil immersion lens to detect

amastigote form of Leishmania donovani. Amastigotes appear as round or oval

bodies measuring 2 to 3 ìm in length and are found in inside or outside the

70

monocytes and macrophages. In preparations stained with Leishman stain, the

cytoplasm appears pale blue, with a relatively large nucleus that stains red. In the

same plane as the nucleus, but at a right angle to it, is a deep red or violet rod-like

body called a kinetoplast. If any amastigote in 1000 fields was found, the slide

was reported as positive. Grading of positive smear was done according to

standard chart.

Score 0 = No LD bodies in 1000 fields

Score 1 = 1-10 LD bodies in 1000 fields

Score 2 = 1-10 LD bodies in 100 fields

Score 3 = 1-10 LD bodies in 10 fields

Score 4 = 1-10 LD bodies per fields

Score 5 = 10-100 LD bodies per fields

Score 6 = 100 LD bodies per fields (DGHS 2009)

3.10 Nested PCR for Leishmania Donovani Kinetoplast DNA (Kdna)

Amplification:

Four major steps:

A. DNA extraction from samples,

B. DNA amplification in thermal cycler,

C. Gel electrophoresis,

D. Visualization / documentation under UV light.

3.10.1 DNA extraction from samples:

1. Buffy coat was taken for DNA extraction

2. DNA was extracted by phenol-chloroform method.

3. 200 ìl buffy coat was homogenized with

-200 ìl lysis buffer [50Mm Tris-HCL (pH7.6),1Mm EDTA]

-10 ìl proteinase K in a 1.5ml microcentrifuge tube.

71

4. The homogenate was then incubated at 370C overnight

5. 200ìl of a phenol-chloroform-isoamyl alchohol mixture (25:24:1by volume)

was added.

6. Mixed gently for 5 minutes and micro centrifuged for 10 minutes at 10,000 rpm

at room temperature.

7. The top (aqueous) phase containing the DNA was removed carefully and

transferred to a new tube. The steps were repeated 1-3 times.

8. An equal volume of chloroform-isoamyl alchohol (24:1) was added. Mixed

gently for 2 minutes and spined for 1 minute at 10000 rpm.

9. Then 300 ìl supernatant, 400 ìl ice cold (100%) ethanol and 10 ìl NaCl was

mixed and kept overnight at -200C or in -700C for 1 hour.

10. It was spined for 20 min in a microcentrifuge at maximum speed and remove

the supernatant was removed.

11. 1ml of (70%) ethanol was added at room temperature.

12. The pellet was dried at 370C temp. for about half an hour.

13. The pellet was dissolved in 20-50 ìl TBE buffer pH 8.0 (10mM Tris HCL,

and 1mM EDTA) (Fakhar et al., 2006).

Preparation of PCR mixture:

1. DDW (double Distilled water) -------------- 38.5 µl, 1 = 38.5 µl

Buffer solution

(Contained 10 mM Tris-HCl, pH 8.3,

50 mM KCl, 1.5mM MgCl2) ------------------------- 5µl, 1 = 5µl

dNTP ---------------------------------------------------- 4µl, 1 = 4µl

Primer (1st pair) ----------------------------------------- 1µl, 1= 1µl

= 48.5µl

Taq Polymerase ------------------------------------------------ 0.5µl

72

In each PCR tube:

Reaction mixture ---------------------------------------------- 49 µl

Extracted DNA -------------------------------------------------- 1 µl

Total volume in each PCR tube --------------------------- = 50 µl

3.10.2 DNA amplification in thermal cycle

Amplification

50 µl amplification mixer was dispensed in thermocycler (Model-Master cycler

personal) tubes. 1.0 µl template was added to each of the tubes and mixed with

pipette tips. Amplification was carried out according to the following directions.

PCR tubes were placed in thermal cycler: Programme was planned

Denaturation at 940C ------------------ 30 seconds

Annealing at 450C --------------------- 30seconds

Extension at 720C --------------------- 1 minute

Final extension at 720C --------------- 3 minutes have been performed

In Nested PCR the amplified product of 1st PCR is used as extracted DNA with

the reaction mixture which contains the 2nd pair of Primer for nested PCR.

Reaction mixture for PCR tubes of 50 µl volume:

DDW (Double Distilled Water) ------------- 38.5 µl, 1 = 38.5 µl

Buffer solution

(Contained 1OmM Tris-HCl, pH 8.3,

50 mM KCl, 1.5mM MgCl2) ----------------- 5µl, 1 = 5µl

dNTP -------------------------------------------- 4µl, 1 = 4µl

Primer (2nd pair) -------------------------------- 1µl, 1= 1µl

= 48.5µl

Taq Polymerase ------------------------------------------------ 0.5 µl

= 49 µl

Cycle repeated for 30 times

73

In reaction mixture there was 2nd pair of primer of was added for Nested

PCR

In each PCR tube:

Reaction mixture ----------------------------------------------- 49 µl

1st amplified product (used as Extracted DNA) ------------ 1 µl

Total volume in each PCR tube ----------------------------- 50 µl

In Nested PCR the following programme is done in thermal cycle:

Denaturation at 940C ------------------ 1`minutes

Annealing at 450C --------------------- 1 minute

Extension at 720C --------------------- 1.5 minute

Final extension at 720C --------------- 3 minutes

3.10.3 Agarose gel electrophoresis

The amplification reaction was visualized on a 2% agarose gel; a 100-bp DNA

ladder (Pharmacia, Uppsala, Sweden) was used as a marker. Samples were scored

as positive when a PCR product of 385 bp could be detected.

3.10.3.1 Preparation of agarose gel

Two percent agarose gel was prepared by melting 1.0 gm agarose in 50 ml 1X

TBE buffer using a microwave oven or gas burner. The melted agarose was

allowed to cool to about 500C and was poured into gel electrophoresis unit with

spacers and comb. After solidification of the gel, the comb was removed and

wells were found.

3.10.3.2 Loading and running the sample into gel electrophoresis

Four µL of PCR product was mixed with 2.0 µL of gel loading dye. The mixture

was slowly by loaded into the well using micropipette tips. Mark DNA of known

size (100 bp ladder) was loaded in one well to determine the size of the PCR

products. Electrophoresis was carried out at 100 volts for 45 minutes.

Cycle repeated for 36 times

74

3.11 Visualization/documentation under UV light

Products were analyzed by electrophoresis in 1% agarose gel containing 0.5,

µg/ml ethidium bromide, in TBE buffer and photographed under UV illumination

(Maury et al., 2005).

3.12 Primers used for PCR in Kala-azar

Target Primer Sequence Tm (0C) Fragment length

Ldl primers_L. donovani

kinetoplast

minicircle

sequene

(SEOID No.1)

(SEQID No. 2)

692 bp

5�-AAA TCG GCT CCG AGG CGG GAA AC-3,

5�-GGT ACA CTCT TCAGTAG CAC-3�

640C

56.50C

PCR product 600bp

385bp 5�-TCG GAC GTG TGT GGA TAT GGC-3� 60.40C

PCR product 385bp

Nested PCR (from Indian study)

592bp 5�-CCG ATA ATAT GTAT CTC CCG-3�

54.60 C

3.13 Immuno-Chromatography Test ICT)

Procedure for antibody detection by ICT as per kit manual

3.13.1 Principle of the test: ICT for Kala-azar is a qualitative, membrane based

immunoassay for the detection of antibodies in Kala-azar in human serum. The

membrane is pre-coated with a novel recombinant Kala-azar (rk39) antigen on the

test line region and chickens anti-protein A on the control region. During testing,

serum sample reacts with the dye conjugate (protein A colloidal gold conjugate),

which has been pre-coated in the test device. The mixture then migrates upward

on the membrane chromatographically by capillary action to react with

recombinant Kala-azar antigen on the membrane and generates a red line.

Presence of this red line indicates a positive result, while its absence indicates a

negative result. Regardless of the presence of antibody to Kala-azar antigen, as the

mixture continues to migrate across the membrane to the immobilized chicken

anti-protein A region, a red line at the control line region will always appear. The

75

presence of this red line serves as verification for sufficient sample volume and

proper flow and as a control for the reagents.

3.13.2 Test Procedure: Serum and Chase Buffer were allowed to reach room

temperature prior to testing. The ICT test strip for Kala-azar was removed from

the foil pouch and 20 µl of serum was added to the test strip in the area beneath

the arrow. The test strip was placed into a test tube so that the end of the strip was

facing downward as indicated by the arrows on the strip. Then 150 µl of the

Chase Buffer solution provided with the test kit was added. The result was taken

within 10 minutes. Background was clear before the result of the test was taken.

Results interpreted after 10 minutes could be misleading.

3.13.3 Results

A) Positive result: The test was positive when a coloured control line and

coloured test line appeared in the test area. A positive result indicated that the

ICT detected antibodies to members of L. donovani complex. A faint line was

considered a positive result. The red colour in the test region will vary

depending on the concentration of anti-leishmanial antibodies present.

B) Negative result: The test was negative when only the coloured control line

was appeared. A negative result indicated that the ICT did not detect

antibodies to members of L. donovani complex.

C) Invalid result: When no coloured lines appeared either at the control line or at

the line areas, the test was taken as invalid. The test was also invalid if no

control line was appeared but a test line was seen, retest using a new test strip

and fresh serum was recommended (Inbios Inc., USA).

76

CHAPTER 4

RESULTS

The present study was conducted on a total of 150 subjects. Among them, 106

were cases of Kala-azar and 44 were age and sex matched healthy controls. Out of

106 cases, 41 were confirmed as kala-azar by detection of LD bodies from bone

marrow aspirates by microscopy and 65 were ICT positive clinically diagnosed

cases of Kala-azar.

Age and sex distribution of subjects is shown in the following table 1. Majority of

the cases (58%) and controls (22) were in the age group of 2-12 years. Number of

cases and controls gradually declined with increase in age. Minimum member of

case (02) and control (01) was in the age group of 57 years and above. Total male

were 63 (59.43%) and female 43 (40.57%) in the cases group and corresponding

values for controls were 26 (59.09%) and 18(40.91%) respectively. Male to

female ration of cases was 1.46:1.

77

Table 1. Age and sex distribution of subjects

Cases (n=106) Control (n=44) Age in Years

Male Female Male Female

2-12 (n=80) 33 (31.13%) 25 (23.58%) 13 (29.54%) 9 (20.45%)

13-23 (n=31) 15 (14.15%) 8 (7.54%) 5 (11.36%) 3 (6.81%)

24-34 (n=20) 7 (6.6%) 7 (6.60%) 3 (6.81%) 3 (6.81%)

35-45 (n=09) 4 (3.77%) 1 (0.94%) 2 (4.54%) 2 (4.54%)

46-56 (n=07) 3 (2.83%) 1 (0.94%) 2 (4.54%) 1 (2.27%)

>56 (n=03) 1 (0.94%) 1 (0.94%) 1 (2.27%) 0

150 63 (59.4%) 43 (40.6%) 26 (59.1%) 18 (41%)

78

Table 2 shows the housing condition of Kala-azar cases. Earthen house and tin

shade were in 68.87% and 31.13% respectively.

79

Table 2. Housing condition among study population

Housing Condition Frequency (N0.) Percent

Earthen 73 68.87

Tinshed 33 31.13

Total 106 100

80

Table 3 Shows Comparison between low and middle socioeconomic status in

Kala-azar cases. Out of 106 Kala-azar cases 64 (60%) were from low economic

status, 36 (34%) were from middle economic status and 6 (6%) were from upper

economic status.

81

Table 3. Comparison between low and middle socioeconomic status in Kala-

azar patients

Socioeconomic status Percentage Percentage Chi-square

Low economic statue 64 60

Middle economic statue 36 34

<0.01**

Legend: ** (p<0.01 level of significant)

Low economic status Income Tk. <5,000 per month.

Middle economic statue Income Tk. (5,000-10,000) per month

82

Table 4 shows Kala-azar cases as per duration of fever. Majority of cases (29.2%)

had fever for 4 months duration followed by duration of 2 months (25.5%) and

duration of 3 and 5 months (18.9%) and (16%). Only 3.8% cases had duration of

fever for >6 months. Mean duration ±SD of fever was found as 3.77±1.32 month.

83

Table 4. Duration of fever in Kala-azar cases

Fever with duration Cases Percentage Mean duration ±SD of

fever in month

2 months 27 25.5

3 months 20 18.9

4 months 31 29.2

5 months 17 16

6 months 7 6.6

>6 months 4 3.8

3.77±1.32

Total = 106 100

84

Enlargement (in cm) of liver (hepatomegaly) and spleen (splenomegaly) of Kala-

azar cases are given in Table 5. Mean enlargement ±SD of liver and spleen was

found as 5.19±2.03 and 6.98±2.58 respectively. Regarding hepatomegaly, highest

enlargement as 12.5 cm was found in (2.8%) and lowest as 2.5 cm in (17%).

Majority (67.9%) cases showed enlargement by 5 cm. Regarding splenomegaly,

highest enlargement as 12.5 cm were found in (4.7%) and lowest as 2.5 cm in

(8.5%). In (34.9%), (30.2%) and (21.7%) cases enlargement were found as 5 cm,

7.5- cm and 10 cm respectively.

85

Table 5. Hepato-splenomegly in Kala azar cases

Enlargement in cm Hepatomegaly (n=106) Splenomegaly (n=106)

2.5 18(17) 9(8.5)

5 72 (67.9) 37(34.9)

7.5 9(8.5) 32(30.2)

10 4(3.8) 23(21.70)

12.5 3(2.8) 5(4.7)

Mean±SD 5.19±2.03 6.9811±2.58

Total 106 106

86

Haematological findings of (41) confirmed cases of kala-azar cases are given in

Table 6. Anaemia, leucopoenia, Thrombocytopenia were found in 34(83%),

18(44%), 33(80.48%) of confirmed cases of kala-azar cases respectively.

Lymphocyte count was found to be above normal level (>45%) in 87.71 %

patients. Erythrocyte Sedimentation Rate (ESR) was raised in 95% of the patients

in this study.

87

Table 6. Haematological findings of (41) confirmed cases of kala-azar

Parameter No. Patients Percentage

Haemoglobin g/dl 8 - 12 7 17.07 4 - 8 28 68.29 <4 6 14.63 Total Leukocyte Count/mm 3 4000 and above (normal) 23 56.9 4000 - 3000 6 14.63 3000 - 2000 8 19.5 <2000 4 9.7 Lymphocyte Count Normal (20 - 45%) 5 12.19 Relative Lymphocytosis (>45% with normal TLC)

21 51.21

Absolute Lymphocytosis (>45%with decreased TLC)

15 36.5

Total Platelet Count Normal (150 - 400×10 9 /L) 8 19.51 Thrombocytopenia (<150×10 9 /L) 33 80.48 Erythrocyte sedimentation rate (mm in 1s hour) <20 2 5 20 - 50 5 12.19 50 - 100 14 34.14 >100 20 48.78

88

Table 7 indicates prevalence of kala-azar cases among study population. Among

41 bone marrow positive confirmed Kala-azar cases, buffy coat microscopy for

LD was positive in 38 (92.7%) confirmed Kala-azar cases and PCR from buffy

coat was positive in 39(95.12%) confirmed Kala-azar cases. Among 65 ICT

positive KA cases, buffy coat microscopy for LD was positive in 60(92.30%) and

PCR from buffy coat was not done. Out of 44 endemic controls, 02(4.54%) were

positive by buffy coat microscopy and 0.00(0.00%) were positive by buffy coat

PCR.

89

Table 7. Prevalence of kala-azar cases among study population

Buffy coat microscopy for LD

PCR from buffy coat Study population (n=150)

Positive Negative Positive Negative

Bone marrow microscopy for LD (n=41)

38 (92.7%) 03 (7.3%) 39 (95.12%) 02 (4.9%)

rk39 positive cases (n=65) 60 (92.30%) 05 (7.7%) 62 (95.38%) 03 (4.61%)

Healthy endemic Control (n=44)

02 (4.54%) 42 (95.45%) 0.00(0.00%) 44 (100%)

90

Table 8 indicates Sensitivity and specificity of LD body in buffy coat of

confirmed Kala-azar cases. Buffy coat microscopy for LD were positive in 38

(92.7%) and were negative in 03(7.3%) of confirmed Kala-azar cases .In controls,

42(95.45%) were negative and 02(4.54%) were positive. Accordingly, Sensitivity

and Specificity were 92.7% and 95.45% respectively.

91

Table 8. Sensitivity and specificity of LD body microscopy from buffy coat in

confirmed KA cases

Test result Confirmed KA cases

Controls Total Sensitivity Specificity

Buffy coat microscopy positive

(a) 38 (92.7%) (b) 02(4.54%) (a+b) 43

Buffy coat microscopy negative

(c) 03 (7.3%) (d)42(95.45%) (c+d) 42

Total (a+c) 41 (b+d) 44 (a+b+c+d) 85

92.7% 95.45%

NB. Sensitivity and specificity was calculated by the following formula

Sensitivity=100×a/a+c , specificity=100×d/b+d

92

Table 9 indicates Sensitivity and specificity of buffy coat PCR in confirmed KA

cases. PCR from buffy coat was positive in 39(95.12%) and were negative in 02

(4.9%) of confirmed Kala-azar cases. In controls, 44(100%) were negative and

0.00(0.00%) were positive. Accordingly, Sensitivity and Specificity were 95.12%

and 100% respectively.

93

Table 9. Sensitivity and specificity of buffy coat PCR in confirmed KA cases

Test result Confirmed KA cases

Controls Total Sensitivity Specificity

Buffy coat PCR positive

(a) 39 (95.12%) (b) 0.00 (0.00%)

(a+b) 43

Buffy coat PCR negative

(c) 02 (4.9%) (d) 44 (100%) (c+d) 42

Total (a+c) 41 (b+d) 44 (a+b+c+d) 85

95.12% 100%

NB. Sensitivity and specificity was calculated by the following formula

Sensitivity=100×a/a+c, specificity=100×d/b+d

94

Table 10 indicates Sensitivity and specificity of LD body in buffy coat of ICT

positive KA cases. Buffy coat microscopy for LD was positive in 60(92.30%) out

65 cases. In controls, 42(95.45%) were negative and 02(4.54%) were positive.

Accordingly, Sensitivity and Specificity were 92.30% (p<0.001) and 95.45%

(p<0.001) respectively.

95

Table 10. Sensitivity and specificity of buffy coat microscopy in ICT positive

KA cases

Test result ICT positive KA cases

Controls Total Sensitivity Specificity

Buffy coat microscopy positive

(a) 60(92.30%)

(b) 02(4.54%) (a+b) 62

Buffy coat microscopy negative

(c) 05(7.7%) (d)42(95.45%) (c+d) 47

Total (a+c) 65 (b+d) 44 (a+b+c+d)109

92.30% 95.45%

NB. Sensitivity and specificity was calculated by the following formula

Sensitivity=100×a/a+c , specificity=100×d/b+d

96

Distribution of kala-azar cases in hospitals are shown in figure 1. Out of 106 kala-

azar cases 71 (66.98%) were from rural hospital (Trishal Thana Health Complex)

and 35 (33.02%) were from urban hospital (Mymensingh Medical College

Hospital).

97

66.98%

33.02%

Rural Hospital (Trishal Thana Health Complex)

Urban Hospital (Mymensingh Medical Collage Hospital)

Figure 1. Distribution of kala-azar cases in hospitals

98

CHAPTER 5

DISCUSSION

Kala-azar is a poverty-related disease. Since the poorest segment of the

community mostly suffer from this disease, so due attention is usually not paid on

behalf of the health authority. Moreover, development researches are also difficult

to perform because of remote and backward approach to reach the endemic

locality. Thereby, it has appeared as a neglected disease, though it has been

constituting a major health problem at regional level in the Indian sub-continent.

It is the essential demand of the time to of control Kala-azar. For this purpose,

timely and adequate treatment may contribute significantly to reducing the human

reservoir of this anthroponotic disease. Chemotherapy is critically important in

reducing the burden of disease and antimonials are the first-line drugs for all

clinical forms. Treatment is long, expensive and not devoid of adverse side

effects. In many states of India, treatment failure is already well documented due

to low responsiveness of parasites to sodium antimony gluconate. In Bangladesh,

resurgence of Kala-azar has been noticed in the decade of 1970 that has gradually

increased to an epidemic form in many areas. Currently Kala-azar appears at a

rate in excess of 15000 per year (Al-Masum et al., 1995; DGHS, 1995). A large

number of studies were carried out mostly on epidemiological, clinical, diagnostic

and therapeutic aspects. In diagnostic aspects, definitive diagnosis of Kala-azar is

a technically difficult job. Because, collection of splenic or bone marrow aspirate

is painful and risky, these samples are not collected or examined at community,

upzila health care center. This study has described the technical aspects of an easy

definitive diagnostic method from blood buffy coat smear and usefulness of

Leishmania nested polymerase chain reaction (Ln-PCR) for diagnosis of kala-azar

99

from the same specimen. We selected majority of Kala-azar cases from Thishal

Thana Health Complex.

In the present study, out of 106 cases, 63 (59.4%) were male and 43 (40.6%) were

female giving a male to female ratio of 1.46:1 (Table 1). Other studies from home

also recorded almost similar ratio between male to female e.g. ICDDRB (2002)

and ICDDRB (2003) found male to female ratio of 1.2:1 and 1.04:1 respectively.

Several local studies were also consistent as showing male to female ratio of

1.38:1 (Sarker et al., 2003), 1.8:1 (Musa et al., 1996), 2:1 (Talukder et al., 2003)

and 2.4:1 (Shamsuzzaman et al., 2003). Similar finding was reported from abroad

where ratio between male to female was 1.3:1 (Antonio et al., 2002). In a study,

from Sudan, it was reported that like many countries males are almost twice

(1.8:1) more likely to be affected by Kala-azar than females (Zijlstra et al., 1994).

Males are infected more often then females, most likely because of their increased

exposure to sandflies due to professional activate (Berman, 1997).

Kala-azar affects various age groups depending on the infecting species,

geographic location, disease reservoir and host immuno-competence. In Indian

type of Kala-azar, children between 5 and 15 years of age are affected more

(Gradoni et al., 1995).

In our study, majority of cases (54.7%) were in the 2-12 years age group (Table

1). Almost similar findings were also found in many studies. Ali and Ashford

(1994) from Ethiopia in a study found 142 cases of Kala-azar, where 58%

children below 15 years. In study by Nuttal et al., (2002) observed that majority

patients of Kala-azar were in the age group of 5-15 years. In one local study on 65

patients, over 60 cases of KA were under 20 years of age (Talukder et al., 2003).

On the other hand, higher number (86%) of KA cases were found under 5 years

children in Pakistan during the period between 1985-1995. This figure was

100

relatively higher in comparison to that of the present study (Rab and Evans,

1995). Higher incidence of kala-azar in children might be due to the reason that

children of poor family generally suffer from malnutrition. Consequently their

immunity is hampered increasing the risk of getting kala-azar infection and

developing severe disease (Desjeux, 2004). Moreover adult population in the

endemic locality might develop protective immunity from previous infection that

reduces the chance of reinfection (Gregory et al., 2008).

In our study, living houses of most (68.87%) Kala-azar cases were made of

earthen walls (Table 2). Due to low socio-economic condition people made their

houses with mud. Easily developed tiny cracks and crevices in the earthen house

are the favourable places for sandflies (Desjeux, 2004). So, people residing in

earthen houses must bear more risk for being infected by KA agents.

In our study, most of the Kala-azar patients 64(60%) were from low

socioeconomic status (Table 3). This finding also correlates with others studies

from Bangladesh who reported that there was a strong association between Kala-

azar and poverty (Bern et al., 2005). Similar findings were also reported by

(Thakur, 2003) and Bern et al., (2000), we also observed the effect of

socioeconomic factors on kala-azar patients.

The extent and presentation of the disease depends on several factors, including

the humoral and cell-mediated immune response of the host, the virulence of the

infecting species and the parasite burden. The remarkable presentation of kala-

azar patients is prolonged low-grade irregular fever associated with organomegaly

of spleen and liver. Anaemia and darkening of skin are also found in majority of

cases (Gradoni et al., 1995).

101

In our study, prolonged low-grade irregular fever was found in 100% cases and

majority of cases (29.2%) had fever for 4 months duration (Table 4). Similar

findings were also reported by other authors both in home and in abroad

describing association of fever in 95-100% cases of kala-azar (ICDDRB, 2003;

WHO, 1996; Maltezou et al., 2000; Sarker et al., 2003). Mean duration of fever in

month of kala-azar cases in the present study was found ad 3.77±1.32. Multiple

authors reported duration of fever in kala-azar cases as over 3 months period

keeping very good agreement with that of ours (Talukder et al., 2003; ICDDRB,

2003; WHO, 1996). The explanation for relatively longer period of fever in kala-

azar cases is well explained by the fact that the onset of fever is usually insidious

with irregular rise and fall. The patient usually does not become prostrated or

anorexic. So, the patient remain ambulatory without hampering physical work.

For this reason, patients remain reluctant to seek medical advice or to be

investigated to elucidate the causes of fever.

In our study, hepatomegaly with mean enlargement of 5.19±2.03 cm and

splenomegaly with a mean enlargment of 6.98±2.58 cm were found in 100% cases

(Table 5). ICDDRB (2003) and Sarker et al., (2003) reported splenomegaly in

100% cases but hepatomegaly in 79% cases. WHO (1996) reported 98%

hepatosplenomegaly from India. Hepatomegaly was found in 85% and

splenomegaly in 99% cases by Maltezou et al., (2000). However, rate of

hepatomegaly obtained in the present study was not showing exact accordance

with other studies. The reason behind this outcome, the delay in part of the

patients came to seek medical advice. Our majority patients attended while they

were in a febrile stage of more than 2 months. The 2 months period is sufficient

enough for development of palpable organomegaly (Oliaro and Bryceson, 1993).

That was why we got splenohepatomegaly in 100% cases.

102

Each of confirmed kala-azar cases (41) showed a high ESR, reduced haemoglobin

concentrations, reduced TLC, increased percentages of lymphocytes in DLC and

reduced TPC (Table 6). Majority of them (68.29%) were having moderate

anaemia. Similar findings have been reported by Rai et al., 2008; Hassan et al.,

1995; Saleem et al., 1986.

In this study, 43.83% patients showed leucopoenia and 52.86% were neutropenic.

Infections were common and severe in these neutropenic patients. A study from

Armed Forces Institute of Pathology in 1986, showed leucopoenia in 50% and

neutropoenia in 100% cases, (Saleem et al., 1986). Similar findings have been

reported by Manzoor et al., 2008.

Lymphocyte count was found to be above normal level (>45%) in 87.71 %

patients. Similar finding was also reported by (Manzoor et al., 2008).

Thrombocytopenia was noted in 80.48% patients in out study. This

thrombocytopenia was the main cause of bleeding in our patients. These findings

about platelet count resemble the findings in the study done by Saleem (1986) in

which 91% cases showed platelet count of <100,000/cmm. Erythrocyte

Sedimentation Rate (ESR) was raised in 95% of the patients in our study. In a

study from Institute of Medical Sciences of Pakistan 1995, ESR was found raised

in 88% of cases.

In the study, out of 150 kala-azar cases, 41 were bone marrow positive cases, 65

were rk39 positive cases and 44 were healthy endemic control (Table 7). Among

these 41 bone marrow positive cases buffy coat microscopy for LD body were

positive in 38 (92.7%) cases and buffy coat PCR were positive in 39 (95.12%)

cases.

103

Out of 65 ICT positive cases buffy coat microscopy were positive in 60 (92.30%)

and PCR from baffy coat were positive in 62 (95.38%).

In this study, in 41 confirmed kala-azar cases, sensitivity and specificity of the

buffy coat microscopy for LD body was found as 92.7% and 95.45% respectively

(Table 8). Almost similar findings were reported by another study done in

Mymensingh region, where buffy coat smear positive result is 92.94% in

confirmed kala-azar cases (Shamsuzzaman, et al., 2007). However, there was no

other local study report to see LD bodies in blood buffy coat smear to compare

with the finding of present study. Williams et al., (1995) made comments that

finding of Leishmania parasite in blood is sometimes possible in paticnt with kala-

azar from Kenya and India.

The present study yielded LD body detection at a relatively higher rate (>92%).

This is well explained by the fact that density gradient centrifugation technique

was adopted in this study using lymphoprep to prepare thick buffy coat in

comparison to that found in wintrobes tubes. As a result more macrophage as well

as more LD bodies were concentrated in buffy coat (DGSH, 2009).

Out of 41 confirmed cases 03 (7.3%) were negative in buffy coat smear may be

due to the lower parasitic load in the circulating blood than in the bone marrow

(Lachoud et al., 2002). However, other reasons, such as temporary may also exist

absence of parasites from the blood of kala-azar cases as reported by Le Fichoux

et al., (1999).

In our study 44 healthy endemic controls, 2 (4.54%) were positive by buffy coat

microscopy. It might be due to asymptomatic carriers (Sundar and Rai, 2002).

The sensitivity and specificity of PCR were 95.12% and 100% respectively as per

shown in Table 9. Different studies found sensitivity and specificity values as

104

follows 97% sensitivity and 100% specificity by Salam et al., (2009), 99% and

100% by Maury et al., (2005). The best sensitivity was obtained in the study

because PCR assay was done in the buffy coat.

PCR assay with buffy coat preparations to detect Leishmania was 10 times more

sensitive then that with whole blood preparation (Sundor and Rai, 2002).

Leishmania as are obligate intracellular parasites (in the vertebrate host) and

therefore are supposedly more concentrated in the buffy coat. On the other hand

Buffy coat prepared with PK-based methods gave the best results (Lachoud et al.,

2000).

Out of 41 confirmed cases 02 (4.9%) were negative by PCR in this study.

Negative PCR results have been reported in a few patients with positive

parasitological proof (Maury, 2005; Cortes et al., 2004).

Low parasitc load could be the cause of less sensitivity of the PCR method (Salam

et al., 2009). In our study, PCR was found negative in only two patients with

grade 1 parasite burden but none of the patients with more then grade 1 parasite

burden was negative by PCR. Therefore, it is reasonable to speculate that negative

PCR might be due to low parasite burden in kala-azar patients. However, other

reasons, such as temporary absence of parasites from the blood of kala-azar cases

as reported by Le Fichouk et al., (1999), cannot be excluded. It is also possible

that inhibitor elements that could be present in the Kala-azar patients sample

might result in negative PCR results.

In this study among ICT positive cases sensitivity of buffy coat microscopy were

92.30% and 95.45% respectively (Table 10). Similar values obtained from buffy

coat smear for LD body among ICT positive cases were reported by

(Shamsuzzaman et al., 2007) in the same institute. Buffy coat smear for LD body

105

were examined among all ICT positive found and some 53 (92.98%) were found

positive (Shamsuzzaman, et al., 2007). Out of 65 ICT positive cases 05 (7.7%)

were negative by buffy coat microscopy. The fact is that an individual with a

positive rk39 strip test result may suffer from an illness (malaria, typhoid fever or

tuberculosis) with clinical features similar to these of kala-azar yet be

misdiagnosed as suffering from kala-azar (Sundor and Ray, 2002).

On the other hand, manufacturer stated that antibody to rk39 antigen could found

in cases of malaria and rheumatoid arthritis (InBios, USA).

Analyzing the findings of the study, it can be concluded that Kala-azar affected

more males than females and majority of them were children under 12 years of

age. In this study explored the reliability and effectiveness of LD body detection

in blood buffy coat smear for definitive diagnosis of Kala-azar and compared the

positivity of the PCR carried out in DNA extracted from buffy coat. The

advantages of performance of PCR on blood buffy coat are non-invasive, reliable,

sensitive and specific.

106

CHAPTER 6

CONCLUSION

Analyzing the findings of the present study, it can be concluded that Kala-azar

remain to be as an endemic disease in this locality. Detection of LD body from

buffy coat by microscopy is more easy, non-invasive and highly sensitive and

specific method. PCR method from same specimen for diagnosis of Kala-azar was

also found highly sensitive and specific.

Thus, efforts should be made to establish LD body detection from buffy coat

smear method at least in the tertiary care hospitals, if possible, at the district-level

hospitals, especially in the endemic areas of Bangladesh and PCR method at least

in the tertiary care hospitals.

107

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140

APPENDICES

APPENDIX I

DATA SHEET

SI.No:

Name: Age: Sex:

Father�s name:

Mother�s name:

Address:

Village: PO: Thana: District:

Hospital Particulars: Regn: Ward: Bed: Unit:

Socio-economic Condition: Poor/ Average.

Housing: Tinshade/ Earthen/ Others. Family history: Positive/ Negative.

Clinical information

Irregular fever: weeks, Emaciation: no/ mild / remarkable

Bleeding: yes / no Appetite: Normal / Decreased /Increased. Jaundice..................

Blackening of skin: yes / not Weight: ........ Anaemia: no / mild /remarkable

Spleen: palpable /not palpable, if palpable: ....cm from left costal margin.

Liver: palpable / not palpable, if palpable: .....cm from right costal margin.

Previous treatment for kA: no/ yes. if yes: Miltefosine/ SAG/ Duration and dose..........

Ongoing treatment:

141

Laboratory information

Haemoglobin: ...... gm/dl.

Platelets: ................... ./cu mm.

TLC: / cu mm of blood.

DLC- N% L% E% M% B%

ESR :..................min in the 1st hour.

Prothrombin time: not done/ sec

BT: not done/ min sec

CT: not done/ min sec

ICT strip: positive/ negative

Spleen aspirate: not done/score

BM: not done / score

Buffy coat: not found / score

Comment:

Signature of data collector

142

APPENDIX II

Making buffy coat (density gradient centrifugation)

In a sterile round bottom disposable test tube (10 ml capacity), 2 ml of Histopaque

pipetted carefully.

Substance Name:

Histopaque 1077

Ingredient

Diatrizoic acid sodium salt dehydrate 9.000%

Ficoll 5.7%

Water 85.300%

Then 2 ml of citrated blood from the vacutainer was poured slowly by the side of the test

tube so that the blood completely overlay the separation fluid without any mixing. The

test tube was centrifuged for 15 minutes. 3000 rpm referable in a swinging centrifuge

machine.

Taking buffy coat material and preparation of smears

After centrifugation, a thick buffy coat was seen in the interface of plasma and

Histopaque. RBCs was settled at the bottom part of the test tube. Now the plasma was

gently sucked by a micropipette of 1ml capacity and was preserved in an eppendrof tube.

Then by another of 100-200 µl capacity/micropipette, the buffy coat material was sucked

out very gently. Part of the material was used in another eppendrof containing 0.5 ml of

95% alcohol for analysis by PCR (DGHS, 2009).

Staining of buffy coat smears

Buffy coat film were stained by Leishman stain.

143

Preparation of Leishman stain:

This stain was prepared by 0.2 gm of powdered dye & was transferred to a conical flask

of 200-250 ml capacity.

100 ml of methanol was added and warmed the mixtures to 500C for 15 mins,

occasionally shaking it

The flask was allowed to cool & filtered. It was then ready for use

Procedure of Leishman staining

A air dried thin film was made and placed on a staining rack and flooded with Leishman

stain. The slide was left for 2 minutes to fix. Double amount distilled water was added

over the flooded stain from a plastic wash bottle for better mixing of the solution. The

stain was left for 10 minutes. The stain was washed with running tap water and air dried

again the smear to examine under oil immersion objectives (DGHS, 2009).

144

APPENDIX III

Equipments for PCR

Centrifuge 5415 R with refrigerator

Vortex Mixer (Digi system VM-2000)

Micropipettee (Company-Eppendrof)

Laminar Flow Cabinet (Company stremline)

Microwave oven for gel preparation

Electric heated dryer (Model Jo2714)

Thermal cycler (Model-Master cycler personal)

Gel apparatus (Major science Minis-300)

Major science Minis UVDI

Preparation of PCR mixture:

DDW (double Distilled water) -------------- 38.5 µl, 1 =38.5 µl

Buffer solution

(Contained 10 mM Tris-HCl, pH 8.3,

50 mM KCl, 1.5mM MgCl2) ------------------ 5µl, 1 = 5µl

dNTP ---------------------------------------------- 4µl, 1 = 4µl

Primer (1st pair/2nd pair) -------------------------1µl, 1= 1µl

= 48.5µl

Taq Polymerase ---------------------------------------------0.5µl

In each PCR tube:

Reaction mixture --------------------------------------------49 µl

Extracted DNA ----------------------------------------------1 µl

Total volume in each PCR tube -------------------------= 50 µl

145

APPENDIX IV

To perform Hemoglobin estimation: Requirements:

Equipments Consumables Refrigerator Colorimeter,

Micropipettes

Gloves, Tissue Paper, Labels, Test tubes � 7/10ml Pipettes � 5/10ml Cuvette Micro tips Measuring cylinder 500ml Drabkin�s solution Hemoglobin control Discard bin & Hypochlorite

Principle:

The method is based on the determination of cyanmethemoglobin

In this method, hemoglobin is oxidized by ferricyanide to methemoglobin.

The methemoglobin is finally converted by cyanide to the stable

cyanmethemoglobin

The concentration of cyanmethemoglobin is directly proportional to

hemoglobin concentration in the blood.

The OD is measured in the Colorimeter at wavelength 540 nm

Procedure

The colorimeter was switched on and adjusted at the wavelength 540.

The samples of blood was arranged to be tested in order and the protocol

sheet was filled in details.

7/10 ml test tubes were labeled with sample number; one tube was

allocated for testing control.

146

5 ml Drabkin�s solution was dispensed to the test tubes, using a pipette and

rubber bulb.

The EDTA tubes containing blood was shacked to ensure thorough mixing

of the cells and plasma.

By using a micropipette and tip, 20ìl of control was added to the test tube

marked �Control.�

This step was repeated till all the samples have been added to the tubes

bearing the corresponding number.

Test tubes were Shacked well (the tube was gently tapped on the palm or

the mouth of the tube was covered with Para film and the tube was

inverted) and was kept at room temperature for 3-5 minutes.

The colorimeter was brought to zero with reagent blank.

The OD of the control solution was taken and then serially the OD of the

samples.

The color intensity was stable for 1 hour; however, the reading was taken

at the same time (either 5 minutes or 10 minutes) for all the patients to

ensure uniformity.

The Optical density value was documented on the protocol sheet

immediately.

147

To perform platelet counts Requirements:

Equipments Consumables

Refrigerator,

Counting chamber

and cover slip

Micropipettes

Gloves, Tissue Paper, Labels,

Platelet diluting fluid, Micro tips

Discard bin & Hypochlorite

Principle:

Whole blood is diluted 1 in 200 in platelet diluting fluid

Platelets are counted microscopically using an Improved Neauber ruled

counting chamber (haemocytometer)

The number of platelets is calculated by adjusting for the dilution and the

volume of fluid used.

Procedure:

The blood was collected up to mark 0.5 from anticoagulated blood

Blood was wiped off from outside the pipette

Platelet diluting fluid was sucked up to mark 11 above

It was then mixed well by rotating the pipette for a few seconds between the

fingers

The cover slip was placed on the counting chamber

The pipette was rotated again for few times and the fluid was discarded

from the stem and a little from the bulb

The tip of the pipette was touched at the edge of the cover slip at about 45º

angle and slowly the fluid was introduced

The chamber was filled by negative pressure

148

Care was taken to ensure that there was no air bubble under the cover slip

The chamber was left undisturbed for 20 minutes for the cells to settle

The chamber was placed under microscope and was focused with 10X

objective.

The cells were then counted with 40X objective in five small squares of the

central 25 square grids.

Platelets were seen as small bright retractile fragments.

149

To perform WBC counts

Requirements:

Equipment Consumables Refrigerator, Counting chamber and cover slip Micropipettes

Manual counter Microscope

Gloves, Tissue Paper, Labels, WBC Diluting fluid, Micro tips Discard bin & Hypochlorite,

Principle:

Whole blood is diluted 1 in 20 in an acid reagent which haemolyze the red

cell, leaving the white cells

WBC is counted microscopically using an Improved Neauber ruled

counting chamber (haemocytometer)

The number WBC per liter of blood is calculated

Procedure:

The blood was collected up to mark 0.5 or 1 either from oxalated or EDTA

mixed blood or directly from a finger prick

Blood was wiped off from outside the pipette

WBC fluid was sucked up to mark 11 above

It was then mixed well by rotating the pipette for a few seconds between the

fingers

The cover slip was placed on the counting chamber

The pipette again was rotated for few times and the fluid was discarded

from the stem and a little from the bulb.

The tip of the pipette was touched at the edge of the cover slip at about 450

angle and the fluid was slowly introduced.

The chamber was filled by negative pressure.

150

Care was taken to ensure that there was no air bubble under the cover slip.

The chamber was left undisturbed for 2-5 minutes for the cells to settle.

The chamber was placed under microscope and the chamber was focused

with 10X objective.

The cells were then counted with 40X objective in four large square

corners.

The cells was counted that touched the upper and right hand margins.

The cells were avoided that touched the left and bottom margins.

151

Determination of ESR

Principal: If blood containing anticoagulant is allowed to stand in a tube, place

vertically. The R.B.C settle down gradually to the bottom since their specific

gravity is greater than that of plasma. The rate in which the R.B.C settle is noted.

Apparatus and reagents:

i) Westergren ESR tube

ii) ESR stand

iii) Disposable syringe with needle

iv) Cotton

v) Test tube

vi) 3.8% sodium citrate

vii) 70% ethanol

Procedure: 0.4 ml of 3.8% sodium citrate was taken in a sterile test tube. Then

1.6 ml of venous blood was added. Test tube was shaken gently to mix the blood

with anti-coagulant properly. Then blood was drawn-from test tube into the

westergren ESR tube upto the mark �O�. Westergren tube was placed vertically by

ESR stand and was allowed to settle down of R.B.C. Then reading was taken at

the end of 1st hour.

152

APPENDIX V

Figure 2. Buffy-coat

Figure 3. Buffy-coat

153

APPENDIX VI

Figure 4. Buffy-coat smear

Unstained buffy-coat smear Leishman stained buffy-coat smear

154

APPENDIX VII

Figure 5. Leishmania donovani in buffy coat smear at 100X (oil immersion) lens

under microscope

Figure 6. Leishmania donovani in buffy coat smear at 100X (oil immersion) lens

under microscope

155

APPENDIX VIII

Figure 7. Leishmania donovani in buffy coat smear at 100X (oil immersion) lens

under microscope

Figure 8. Leishmania donovani in buffy coat smear at 100X (oil immersion) lens

under microscope

156

APPENDIX IX Figure 9. PCR amplification product of Leishmania donovani from buffy coat

samples, Lane MW = 100 bp DNA ladder, Lane 1-15 patients buffy coat

sample. Products were analyzed by electrophoresis in 1% agarose gel

containing 0.5 g of ethidium bromide/ml in TBE buffer and

photographed under UV illumination.

1 2 3 4 5 6 MW 7 8 9 10 11 12 13 14 15 MW

400bp 300bp 200bp 100bp

385bp

157

Figure 10. PCR amplification product of Leishmania donovani from buffy coat

samples, Lane MW = 100 bp DNA ladder, Lane 1-8 patients buffy coat

sample; Lane NC = Negative control. Products were analyzed by

electrophoresis in 1% agarose gel containing 0.5 g of ethidium

bromide/ml in TBE buffer and photographed under UV illumination.

MW 1 2 3 4 5 6 7 8 NC NC MW

400bp 300bp

200bp

100bp

385bp

158

APPENDIX X

Figure 11. rK39 immunochromatographic dipstick test for kala-azar

The test is considered positive with the appearance of two red lines (one in the control area and another in the test area)

The test is considered negative with the appearance of a single red line in the control area

159

APPENDIX XI

Figure 12. Bone marrow smear showing amastigote form of Leishmania

donovani

160

APPENDIX VI

STATISTICAL FORMULA Formula for mean

-

x = n

x

-

x = mean of observations

x = individual observations

n = number of observation

Formula for Standard Deviation

SD = 1

)x -(x 2-

n

SD = Standard deviation

(n-1) = applicable for sample -

x = mean of observations

x = individual observations

n = number of observation

Formula for Chi-square test (2):

2=

E

E) - (O 2

Here,

O = Observed frequencies

E = Expected frequencies

df = Degrees of freedom = (c-1) (r-1)

Operational definition:

Persons living in a family having a per capita income per month of less than 1500

TK. were considered as low economic status.