The Effect of a New Compound (Ravinol Salts) and Yemeni Honey on Cutaneous Leishmania Parasites and Skin's Microbes

Contents

Title

Acknowledgement

Contents

List of table

List of figures

List of abbreviation

Abstract

Chapter I.

I. 1. INTRODUCTION
I. 2. THE AIM OF STUDY

Chapter II.

2. Literature review

2.1. Leishmaniasis
2.1.1. History of Leishmaniasis
2.1.2. The Taxonomy of Leishmania
2.1.3. Life cycle of Leishmaniasis
2.1.4. Morphology of Leishmania parasite
2.1.5. Pathogenesis and Immunity
2.1.6. Epidemiology and Ecology of Leishmaniasis
2.1.7. Clinical symptoms of Leishmaniasis
2.1.8. Diagnosis of Leishmaniasis
2.1.8.1. Direct visualization or isolation of the parasite
2.1.8.2. Culture examination
2.1.8.3. Isolation and inoculation in experimental animals
2.1.8.4. Immunological methods of diagnosisof CL
2.1.8.5. Molecular methods
2.1.9. Treatment of Leishmaniasis
2.1.9.1 Antimony Compounds
2.1.9.2 Amphotericin B
2.1.9.3 Miltefosine
2.1.9.4 Pentamidine
2.1.9.5 Paromomycin
2.1.9.6 Azoles
2.1.10. Prevention
2.1.10.1. Vector control measures
2.1.10.2. Chemical control
2.1.10.3. Environmental control
2.1.10.4. Biological control
2.1.10.5. Role of community participation in sandfly control
2.1.11. Vaccins
2.2. Honey
2.2.1 History of honey
2.2.2 Medicinal properties of honey
2.2.2.1 Antibacterial activity of honey
2.2.2.2 Treatment gastroenteritis by honey
2.2.2.3 The use of honey as wounds and burns dressing
2.2.2.4 Honey and oral health
2.2.2.5 Treatment of cutaneous leishmaniasis by honey
2.3. Rivanol salts
2.3.1. Acridines as antiprotozoal drugs

Chapter III.

3. MATERIALS AND METHODS

3.1. Parasitological analysis
3.1.1. Patients and study area
3.1.2. Blood Collection and Cases Preparation
3.1.3. The Interview with the patients
3.1.4. Collection and examination of skin scrapings
3.1.5. Leishmania Cultivation
3.1.6. Animal inoculation
3.1.7. Examination of samples
3.1.8. Hematological tests
3.2. Microbiological analysis
3.3. Rivanol compounds simple
3.3.1. Test compounds, salt of 2-ethoxy-6,9-diaminoacridne with oxamoylaminoacids.
3.3.2. Preparation of the 2-ethoxy-6,9-diaminoacridinium 3,4-
dimethylphenyl-α-alaninate.
3.3.3. Aspects of Using Honey on Cutaneous Leishmaniasis
3.3.4. Antibacterial study
3.3.5. Determination of minimum inhibitory concentration (MIC) of different brands of honey
3.3.4. Application with RIV and Honey (Sider and Summar)

Chapter IV.

4. RESULTS

4.1.Types and distribution of lesions
4.2. Results of experimental animal
4.3.6. Hematological picture
4.5. Results of antibacterial activity of two different Yemeni honeys (Sider and Summar)
4.6. In vitro results of antimicrobial effects of the RIV
4.7. In vitro antimicrobial effects of the maxis of RIV with Yemeni Honey (Sider and Summar)

Chapter V.

5. DISCUSSION

6. CONCLUSION

7. RECOMMENDATION

8. REFERENCES

Acknowledgements:

In the beginning, thanks to Allah for helping me to allover this work. I cannot find words to express my feeling towards my supervisors for this great help and extra direction and advice in producing this thesis. My gratitude and especial thanks go to: Dr. Khaled Nasher Qhatan associate prof., of Microbiology, Department of Biology, Collage of Education-Radfan, Aden University, Republic of Yemen. I would like to thank. Dr. Khaldoon Mohammed Al-rahawi Prof. of Medicinal Chemistry department Sana‘a University for his extereme effort to complete this work .

My sincere gratefulness to: Dr. Nagat Ali Muqbil associate prof. of Parasitology, and the head of biology department, Faculty of Aden Education for all facilities in this work. I would like to thank Dr. Omar Bin Shuaib. Alos i would like to thank my dear friend Mr. Mohsen Al-Gafery manger of health office Al-Melah district Lahj Governorate .

Mansoor/ 2013

List of Tables

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List of figure

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List of abbreviations

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Abstract

Cutaneous leishmaniasis is still a major health problem especially in countries with low socioeconomic development. Although pentavalent antimony compounds are still the expensive, standard, basic and first line of treatment in Yemen. The aim of study: Detection of Leishmania spp. patients. Detection of other microbes. Study the antiseptic effect of different concentrations of the 2- ethoxy-6,9-diamino acridinium

enzensulphohydrazidooxalyl glycinate and Yemeni Honey (RIV) on a particular strains of microbes in vitro and Leishmania parasite in vivo.

The samples have been collected at the laboratory of the Faculty of Education-Radfan , Aden University. The Cutaneous Leishmaniasis diagnosed clinically parasitology, hematology, and culturing the parasites and biochemically. Moreover, the microbes which are found around the lesions were diagnosed by microscopic, culture and biochemical tests. The venous blood have been collected from 8 patients and 5 controls. The susceptibility of the microbes for some antibiotic found in our pharmacies and the new compound 2- ethoxy-6,9-diamino acridinium benzensulphohydrazidooxalyl glycinate have been studied. Besides, the effect of Yemeni Honey (Sider and Summer) has been studied. Moreover, the animals have been injected with cutaneous leishmaniasis parasite have been studied.

The study has revealed that the age of patients is between 12- 68 years and 4 of them (50%) are males and the other 4(50%) are females. Also the study has showed that most of the patients are young adults live in small villages.

The study has showed that the lesions of 62.5% of the cutaneous leishmaniasis patients are localized on the face, while the lesions of 20% of the patients are in the hands and one case on leg. Besides, it has noticed that all the inoculated rabbits after two-three weeks could develop different types of lesions ranging from nodules to open ulcerated lesions. The smears taken from the patients have showed that are intracellular amastigote, inflammatory cells, lymphocytes, and plasma cells. The amastigote has been found intracellular as well as extracellular.

The results of the experimental treatment showed a good response to treatment after one week with topical dressing of a mixture of natural raw Yemeni honey (Sidr honey) and RIV and the other two rabbits with Summer honey and RIV. The healing of the experimental animals is completely achieved three weeks later. Concerning the first rabbit, the results showed a good response to treatment after one week, but healing was completely three weeks later, concerning the second rabbit, after two weeks of treatment, but healing was completely three weeks later achieved after five weeks. The others group. They treated by using the RIV daily the healing was completely achieved above eight weeks.

The two kinds of Honey have showed a spectrum of antibacterial activity with their growth inhibitory effect against at least two-three different bacterial species including (Citrobacter freundii, E. coli, Klebsiella spp., P. aeruginosa, Staphylococcus aureus and β-Hemolytic streptococci). Besides, it was revealed that all the six isolated tests were sensitive to a 100% v/v solution of Yemeni (Sider and Summer) honey in an agar (well and disc) diffusion assay, but none showed sensitivity to a 25%, 50%, and rarely 75% solution of honey.

The effects of topical dressing of mixture Yemeni natural raw honey (Sider and RIV) in vivo used in resorption therapy have been investigated in an experimental model of cutaneous leishmaniasis of animals. Acridine RIV and mixture treatment of disease cutaneous leishmaniasis in rabbits as evidenced by long-term disappearance of lesions and disappearance of amastigotes in lesion sites and as determined by microscopically analysis and cultivation of material obtained from lesions. Acridine RIV is, therefore, a new lead compound for the synthesis of drugs effective against cutaneous leishmaniasis and Leishmania spp. Acridine RIV is somewhat active like honey and it is more active when is mixed with honey. Besides, Acridine RIV is active against five strains of bacteria (Citrobacter freundii, E. coli, P. aeruginosa, Staphylococcus aureus and β-Hemolytic streptococci.).

The above results show that in microbiology and clinical tests, honey offers advantages in controlling the bacteria growth and in the treatment of certain Health problems. The topical dressing of cutaneous leishmaniasis ulcers with honey is very effective and helps in the treatment in a short term as compared with RIV treatment. However, honey and RIV are more effective together than if they used alone.

The efficacy of honey and RIV in the treatment of cutaneous leishmaniasis is an area for further studied.

1.1. Introduction

The leishmaniases are a group of diseases caused by infection with protozoan parasites of the genus Leishmania. The infection is transmitted by bites from sand flies infected with the parasite (Desjeux, 1996).

Leishmaniasis has two main clinical presentation forms (cutaneous and visceral), which are associated with a broad range of signs, symptoms and degrees of severity (Reithinger et al., 2007). Of the 88 endemic countries, 22 are in the New World and 66 in the Old World with an estimated incidence of 1-1.5 million cases of cutaneous leishmaniasis (CL) and 500, 000 cases of visceral leishmaniasis (VL) (Singh, 2006). Overall prevalence is 12 million people and the population at risk is 350 million (Desjeux, 2004 and Murray et al., 2005). CL is still a large world problem leishmaniasis, is one of the major health problems of the world (Hepburn, 2000) and about 1 to 2.5 million new cases are reported annually (Murray et al., 2005). CL is a zoonotic disease caused by protozoa of the genus Leishmania and is transmitted to humans.

The clinical outcome will depending on the parasite strain and the host immune response. The lesions are confined to the skin and to the mucous membrane a granulomatous response occurs and a necrotic ulcer forms at the bite site. Macrophages containing amastigotes, which may be killed by sensitized lymphocytes were detected in microscopic smear. The lesion may become chronic, usually accompanied by secondary bacterial infection (Paul and Junk, 1985). These scars have deep psychological effects on the patient and could decrease their career opportunities (Davies et al., 2000a).

Treatment in single lesion it may be injected into the margin of the ulcers. Leishmaniasis responds poorly to treatment and frequently relapses. Secondary infections add to the severity of lesions both in the skin and mucous membranes (Zenia et al., 1996). Antimonial compounds have been used as the first choice drugs for the treatment of leishmaniasis for more than 50 years, but there is still a huge gap to the ideal treatment for this disease (Croft and Coombs, 2003). Problems of injection, side effects, drug resistance and treatment failure have made pentavalent antimony compounds not favorable in the treatment of CL (Masmoudi et at., 2006 and Rojas et al., 2006).

The problem is further aggravated by the appearance of resistance to these drugs in some endemic areas. Amphotericin B and pentamidine are second-line drugs and they present limited value because of their toxicity and difficulty in administration (Berman, 2003). As a result of over-use and abuse of antibiotics there has been an increase in the number of diseases, which seem to evolve to become more virulent with each generation.

Acridine derivatives are one of the oldest and most successful classes of bioactive agents (Denny, 2002). Rivanol salts (acridines) as antimicrobial agents was first proposed by Ehrlich and Benda in 1912, and the first clinical use of these agents occurred in 1917 (Morgenroth et al., 1921). Many compounds containing the acridine chromophore were synthesized and tested, and the aminoacridines found wide use, both as antibacterial agents and as antimalarials, during World War II. The emergence of the penicillins eclipsed the acridines in antisepsis due to the greater therapeutic efficacies of the former (Molten et al., 1999). However, with the current massive increases in drug-resistant microbial infection, new acridine derivatives may be of use. In addition, the topical utilization of aminoacridines in conjunction with directed low-power light offers microbicidal action at much lower doses.

Investigations into natural and potent antimicrobials seemed to be the right step to take. The invasion of pathogenic organisms is on the rise. Efforts are being made to develop antimicrobial agents from natural sources for better therapeutic effects (Gills, 1992). Due to the side effects and the resistance that pathogenic microorganisms have developed against antibiotics, recently much attention has been paid to extracts and biologically active compounds isolated from natural species used in herbal medicine. The antibacterial activity of honey was first recognized in 1892 (Dustmann, 1989).

The use of alternative therapies is mostly due to development of antibiotic resistance in bacteria and/or increasing awareness on the adverse side effects of many pharmaceuticals (Fearnley, 2001). Antibiotic resistance emerged as major global problem (Amabile-Cuevas, 2006).

Various characteristics of honey in destroying a wide range of microorganisms (Cooper, 2008 and Maeda et al., 2008), its anti-inflammatory effects (Van den Berg et al., 2008) and its significant effect in healing wounds (Abdelatif et al., 2008 and Betts, 2008) have led to the use of this natural product in several medical fields (Khan et al., 2007). Actually honeys vary according to their plant origin and the conditions of their production (Bogdanov, 1997). However, it has a limited use in medicine due to lack of scientific support (Ali et al., 1991). The precise mechanisms of action are still not fully understood, however, the antimicrobial activity of most honeys is linked to the production of hydrogen peroxide by the enzyme glucose oxidase which, combined with high acidity, exerts an antimicrobial effect (French et al., 2005). In addition, unidentified phytochemical factors (non-peroxide factors) exert a high antimicrobial effect in some honeys (e.g. Manuka honey) that do not breakdown when treated with heat or light and are still effective even when diluted (Olaitan et al., 2007).

1.2. The aim of study

This study aimed to the following :

1. Detection of Leishmania spp. patients.

2. Detection of other microbes accombined with leishmania.

3. Study the antiseptic effect of different concentrations of the compounds Rivanol salt and Yemeni Honey on a particular strains of microbes in vitro and Leshmania parasite in vivo.

2. Literature review

2.1. Leishmaniasis

2.1.1. History of Leishmaniasis

Leishmaniasis is considered by the WHO to be a neglected emerging disease and one of the most important parasitic diseases with 10% of the world population at risk, (Cruz et al., 2006). Leishmania is an intracellular protozoan parasite belonging to the family Trypanosomatidae, genus Leishmania (Roberts, 2006). The leishmaniases are caused by twenty species considered pathogenic to humans (WHO, 2009). These organisms fall within two main groups, the Old World species occurring in Europe, Africa and Asia and the New World species occurring in the Americas (The Center for Food Security and Public Health, 2009).

Old World CL, known as oriental sore, is an ancient disease and can be traced back many hundreds of years. There exist records of what seems to be CL at least as far back as 650 BC, and possibly much earlier in the Tigris/Euphrates basin (Dedet and Pratlong, 2003). The origins of leishmaniasis are unknown (Momen and Cupolillo, 2000). There are records of lesions similar to the ones caused by leishmaniasis on tablets from the library of Ashurbanipal, King of Syria, from the 7th century BC. These are thought to be derived from earlier texts from 1500 to 2500 BC (Cox, 2002).

Leishmaniasis has been an antique public health problem in South-West Asia and the Arab World reported from time immemorial as the pharaohs ruled in Egypt and Assyrians in Mesopotamia. It was extensively described by Arab- Islamic scientists like Avicenna (Ibn- Sina, 980-1037 AD), who wrote a complete chapter in his prominent book entitled Alkanoun Fi El Tebb raising the possibility of mosquitos being involved in the transmission of the disease. Al Rhazi (AD 850 to 923) already described CL as a disease endemic in Balk (Afghanistan) and Baghdad and in Aleppo-Syria in 1756 (Cox, 2002).

In the Old World leishmaniasis has a long history as descriptions of CL are found since the first century AD. Similarly in the New World pottery from Ecuador and Peru dating from 400-900 AD illustrates faces afflicted with a process consistent with leishmaniasis (Choi and Lerner, 2001). The first descriptions in English of a lesion resembling leishmaniasis was made in 1756 by Russell who described the "Aleppo evil", from Syria. In 1885, Cunningham observed organisms in macrophages from lesions of "Delhi boil" in India (Choi and Lerner, 2001). He found nucleoid bodies of equal size clustered in mass. He thought they were spores and thus postulated that the Delhi boil had a fungal origin.

In 1898, a Russian military sergeant Borovsky reported from the Tashkent military hospital that bacterial agents described in Start sores were artifactual and the actual causative organism was a protozoan and described the anatomy of the organism and pointed out the kinetoplast. He described in detail these parasites in cases of CL, but he did not name them (Hoare, 1938). In 1903 Leishman published his finding of the parasite in spleen of a patient who had died of Dumdum fever in Dumdum, India in 1900. A few months later Donovani described identical organism in a spleen aspirate from a living child. Ross named the parasite as "Leishmania donovani". There were many other names for leishmaniasis include oriental sore, Aleppo evil, Delhi boil, Baghdad sore, Rose of Jericho, Chicler‘s ulcer, uta, espunda (mucous form), forest yaws, Dumdum fever (Visceral form), Kala azar and black fever (Choi and Lerner, 2001).

Cutaneous leishmaniasis is an endemic disease in Yemen (Khatri et al.,2006). Unfortunately, there is no definite treatment for this disease (Nilforoushzadeh et al., 2007). In the poorer suburbs of Middle Eastern Countries such as Afghanistan, Iran, Turkey and Syria, population density is high and sanitary conditions are poor, providing ideal breeding grounds for sandflies. As a result there has been a progressive increase in the number of cases of CL reported from Aleppo in Syria. It is estimated that approximately 60% of dalys lost due to tropical-cluster diseases prevalent in Yemen (WHO, 2008).

The first reported case of CL in African countries was in Tunisia in 1903 and Ethiopia in 1913 and the first case of Visceral leishmaniasis (VL), was reported in Sudan in 1904 (Oumeish, 1999). There was also additional evidence that indicated the first observed case of ulcerative CL in Ethiopia was in 1912 by Martoglio while Balazer and others reported the first non-ulcerative CL in 1960 from the highland area, and likewise the first report of VL in Ethiopia was from Omo Rate and Kelem at the North-West end lake Turkana in 1926 (Humber et al., 1988).

2.1.2. The Taxonomy of Leishmania

Various forms of clinical manifestations of human leishmaniosis have been described and divided into three entities: visceral leishmaniosis (VL, kala azar), cutaneous leishmaniosis (CL, oriental sore, uta, pian bois, chiclero‘s ulcer) and mucocutaneous leishmaniosis (MCL, espundia) (WHO, 1990). In the New World1, leishmanioses are caused by L. braziliensis complex (MCL and CL), L. mexicana complex (CL), L. peruviana (CL) and L. infantum (VL and CL); in the Old World, the aetiological agents are L. donovani (VL), L. infantum (VL and CL), L. tropica (CL), L. major (CL) and L. aethiopica (CL). Leishmania infantum and L. chagasi have been found to be identical by biochemical genotyping and should be regarded as synonyms (Mathis and Deplazes, 1995). The diseases are mainly zoonoses with two exceptions, that of CL due to L. tropica in urban areas of Near and Middle East, and that of VL due to L. donovani the Indian sub-continent (northern India, Nepal and Bangladesh). Canine leishmaniasis (CanL) is a chronic viscero-cutaneous disease caused by L.i n f an tu m (= L. chagasi), for which the dog acts as the source reservoir. In some instances, parasites belonging to L. braziliensis complex , L. major and L. tropica have been isolated from this host (Ryan et al., 2003). Leishamania is one of protozoan parasites ground in the order of Kinetoplastida and in the family of Trypanosomatidae (Fig 2.1.2.1)

Abbildung in dieser Leseprobe nicht enthalten

Figure 2.1.2.1. Taxonomy of Leishmania species (Bañuls et al., 2007).

2.1. 3. Life cycle of leishmaniasis

There are two important stages in the life cycle of Leishmania: amastigotes found in man and other non-human reservoir mammals and promastigotes found in the sand fly (Fig 2.1.3.1.). In the vertebrate host. CL is caused by the amastigote form, which infects the macrophage cells in which it multiplies by binary fission, causing death of the host cell. The lesion then becomes necrotic (UI Bari and Rahman, 2008).

Leishmania parasites‘ life cycle is complex. Specifically, these parasites have two basic life cycle stages: one extracellular stage within the invertebrate host (phlebotomine sand fly) and one intracellular stage within a vertebrate host. The parasites exist in two main morphological forms the amastigotes and promastigotes, which are found in vertebrate and invertebrate hosts, respectively (Koutis, 2007). The invertebrate hosts are small insects of the order Diptera, belonging to the Phlebotominae subfamily and only two of the six genera described are of medical importance: Phlebotomus of the Old World (Africa, Asia and Europe) and Lutzomyia of the New World (the Americas) (Killick- Kendrick, 1999).

Some phlebotomine species such as Phlebotomus papatasi and P. sergenti can support the growth of only those species of Leishmania with which they are infected in nature, whereas other species such as Lutzomyia longipalpis and P.argentipes can develop mature transmissible infections when infected with several Leishmania species (Koutis, 2007 and Rogers et al., 2004). These parasites are obligatory intracellular and invade neutrophils (Peters et al., 2008), and dendritic cells (DC) soon after promastigotes into the host‘s dermis. In MØ, the promastigotes transform into amastigotes in a phagolysosome termed as parasitophorous vacuole. In this vacuole, the amastigotes reside and multiply by binary fission until they fill and rupture the cell.

There is an assumption that amastigotes also reside in the cytoplasm compartment may leave the cells through a mechanism that would resemble excoytosis (Rittig and Bogdan, 2000). Latter amastigotes will be taken up by the sand-fly along with the blood cells during her blood meal on an infected host. After ingestion in the sandfly the parasite turns into a promastigote, in the gut of the sand-fly, and then attach to the midgut epithelium of the fly and divide by binary fission (Sacks, 2001). A flagellum appears which is 1 μm in size to begin with and then grows to a full length in 4 hours (UI Bari and Rahman, 2008). These promastigotes then become differentiated into infective metacyclic promastigotes, which are able to detach from the gut and reach proboscis by fast-swimming towards the pharynx of the sand-fly (Bañuls et al., 2007). The sandfly can than transmit the parasite to a new host during her blood meal by introducing metacyclic promastigotes into the dermis and the parasite life cycle will be completed there is also a possibility of transmission of leishmaniasis through blood transfusion and it has been proposed that much of VL associated with human immunodeficiency virus (HIV) infection in southern Europe is transmitted by sharing contaminated needles and syringes during misuse of druges (Ashford, 2000). The potential diseases reservoir include many different orders of mammals such as rodents, canids, edentates, marsupials, procyonids, primitive ungulates and primates. Human beings are usually accidental hosts of leishmaniasis, because they live in endemic zones and are thereby exposed to infected sandflies (UI Bari and Rahman, 2008). Transmission of leishmania infection occurs almost exclusively through the bite of an infected sandfly; however, other possible modes of transmission reported are the direct transmission via skin contact of CL and congenital transmission in VL. The exact mode of transmission of parasite from the vectors mouth to man is not very clear because the sandfly may bite many times but may not always transmit the disease even though it may be heavily infected (Grevelink and Lerner, 1996) .

Abbildung in dieser Leseprobe nicht enthalten

Fig 2.1.3.1. Life cycle of human cutaneous leishmaniasis (UI Bari and Rahman, 2008).

In a successful bite, between 10-200 promastigotes enter the dermis during each feeding by an infected sandfly (Farah et al., 1993). Parasites on entering the body through the bite of the sandfly are introduced into the blood stream. There they encounter complement, antibodies, and phagocytic cells. All of these can kill the promastigotes. Almost 80% of these are killed one way or the other. Survivors are found in the phagocytic cells, both in neutrophils and mononuclear phagocytic cells. Different factors contribute to the attachment and uptake of parasites into the host cells, long-term survival is possible only in the macrophages. For the disease to be initiated the promastigote must enter the mononuclear phagocyte evading the various potentially lethal cells and humoral factors. Since the macrophage is equipped with mechanisms that are destructive for the parasite, therefore the parasite must possess factors that reduce the impact of macrophage destructive mechanism. The initial interaction with the macrophage membrane may influence the fate of the parasites once they are taken in the cells (Wright and El-Amin, 1989).

2.1.4. Morphology of Leishmania parasite

The parasite Leishmania exists at least in two forms

Amastigote form: Amastigote are small, exist in mammalian host cell and are rounded, non-flagellated and measures 3-5µm in size. Amastigotes predominately exist and multiply in the mononuclear cells such as MØ (Sack and Kamhawi, 2001). They are colourless, have a homogenous cytoplasm and are surrounded by a pellicle. The nucleus is centrally located anterior to which is the kinetoplast. The kinetoplast is a section of the mitochondrion in which the mitochondrial DNA is arranged in regular arrays of fine fibrils (Chang et al., 1985). On simple light microscopy, a central round or oval nucleus and adjacent but smaller round or rod shaped kinetoplast can be discovered. An infolding of the surface membrane creates an internal space, termed as ‗flagellar pocket‘.

The flagellum is not functional in amastigotes and does not extend beyond the cell body. In addition to anchoring the flagellum the main function of the pocket is to function as a site of endocytosis and exocytosis (Webster and Russel, 1993). Immediately below the origin of the flagellum lies a dense mass of mitochondrial DNA known as kinetoplast. The kinetoplast DNA is composed of several thousand circular DNA molecules linked together in a catenated network (Shlomai, 1994). These DNA networks are of two types: each kinetoplast contains 25-250 maxicircles of approximately 30kb, and 5000-10,000 minicircles of about 2kb size each. Together these constitute the mitochondrial genome. The cytoplasm contains both rough and smooth endoplasmic reticulum. The Golgi complex is typically found in the vicinity of the flagellar pocket, which probably reflects the role of this organelle in the endocytic and exocytic pathways. Lysosomes are also found in the cytoplasm together with an organelle unique to kinetoplastids, the glysosome (Opperdoes et al., 1991).

The developmental cycle is initiated by the interaction of metacyclic promastigotes with skin macrophages. After uptake and internalization of metacyclic promastigotes in a phagosome, fusion with lysosomes proceeds as normal and the parasite inhabits a secondary lysosome or phagolysosome. During this process the metacyclic promastigote transforms into an amastigote within 12-24 h and continues to grow and divide within the phagolysosomal compartment. The amastigotes have to overcome two environmental challenges: the battery of lysosomal enzymes and low pH (4.5-5.5). Low pH is not a problem as amastigotes seem to be acidophiles: they are metabolically more active at low pH (Zilberstein and Shapira, 1994).

P romastigote form: In the sandfly host the parasite is found in the promastigote form. The transformation of amastigotes to promastigotes starts within hours of ingestion of the amastigotes (either free or intracellular) and occurs exclusively in the gut. The amastigotes are completely transformed into motile promastigotes within 24-48 h and keep on dividing by binary division. The mature metacyclic promastigotes are accumulated in the midgut and foregut (Herwaldt, 1999). Promastigotes exist in vitro culture at 25˚C and are morphologically elongated flagellated and measures about 10-15µm (Sack and Kamhawi, 2001).

There is a variety of different promastigote forms that can be separated on morphological grounds but functional distinction is less complete (e.g., procyclic promastigotes, paramastigotes, nectomonad promastigotes, haptomonad promastigotes, paramastigotes and metacyclic promastigotes). The first developmental event in the sandfly is probably the transformation of amastigotes to procyclic promastigotes. These events occur in the posterior midgut of the sandfly (Ashford and Bates, 1998). Multiplication of procyclic promastigotes occurs, they elongate and transform to nectomonad forms of 15-20 μm body length. Approximately 3 days after bloodfeeding the peritrophic membrane (a secretory sheath) which contains these parasites usually begins to breakdown and promastigotes begin to set free and they forward to the anterior midgut (Ashford and Bates, 1998).

2.1.5. Pathogenesis and Immunity

Leishmanias are obligatory intracellular protozoal parasites of the genus Leishmania, family Trypanosomatidae. There are more than 17 Leishmania species known to cause infection in humans. VL is caused by the Leishmania donovani complex, which includes L. donovani in the Indian subcontinent, Asia, and Africa, L. infantum in the Mediterranean basin, and L. chagasi in South America (Murray et al., 2005 and Croft et al., 2006).

Leishmanias remain within sandflies with the extracellular promastigote flagellate form. Following a sandfly bite and inoculation into the skin of the host, leishmanias are phagocytized by dermal macrophages where they transform into the intracellular amastigote form (Murray et al., 2005 and Chappuis et al., 2007). Leishmanias replicate and disseminate to additional local or distal macrophages in the reticuloendothelial system of the host, resulting in infiltration of bone marrow, liver, spleen, and lymph nodes (Murray et al., 2005; Chappuis et al., 2007 and Santos et al., 2008).

Immunity against leishmanial infection is mainly T-helper cell-type 1(Th1)-depended (Chappuis et al., 2007 and Antinori et al., 2008). Following phagocytosis by macrophages, immune responses are triggered. The maintenance of macrophages in a deactivation stage mainly determines progression of intracellular leishmanial infection and further dissemination. Non-specific and antigen-specific (cell-mediated) immune responses mediate clinical expression of leishmanial infection and prevention of reactivation, and also determine responses to antileishmanial treatment (Murray et al., 2005). There is no protection conferred by specific antibody responses.

Interleukin 12 (IL12) promotes cell-mediated immunity against leishmanial infection. Activated CD4 T cells are recruited to cutaneous or visceral sites of infection and drive inflammatory responses at the local level, including the localized formation of granulomas and lesions. CD4 T-cell immune responses are associated with interferon-ᵧ (INF- ᵧ)- induced macrophage activation and increased production of cytokines, mainly IL12, but also IL2 and tumor necrosis factor. INF- ᵧ production and activation of CD4 T-cells also promote CD8 T cells participation (Murray et al., 2005 and Antinori et al., 2008). Deactivation of macrophages with subsequent dissemnination of intracellular leishmanial infection is induced by Th2 cytokines, namely IL4, IL10, and IL13. Uncontrolled leishmanial infection resulting in clinical manifestation of VL is likely maintened by IL10 production, whereas IL10 neutralization has been associated with activation of Th1 response and leishmania killing (Murray et al., 2005 and Chappuis et al., 2007). Most of these observations regarding cellular responses and cytokines have been made in murine models (Antinori et al., 2008 and McFarlane et al., 2008). Analogous data on cytokines expression have been found in humans with Leishmania infections (Chappuis et al., 2007). Delayed-type hypersensitivity is maintained in asymptomatic leishmanial infection, remains negative in untreated VL, but reverses to positive several months following successful treatment, as shown by skin leishmanin tests (Murray et al., 2005). However, once leishmanial infection has been acquired, leishmanias persist intracellularly in the skin throughout lifespan, even following spontaneous recovery or successful treatment, representing a potential source for relapse if protective Th1 immune mechanisms fail (Chappuis et al., 2007). Recent data show that uncontrolled infection (as is the case in untreated typical VL) may be determined by genetic factors favoring a Th2-type than a Th1-type immune response (Murray et al., 2005; Chappuis et al., 2007; Jeronimo et al., 2007 and McFarlane et al., 2008).

2.1.6. Epidemiology and Ecology of leishmaniasis

Cutaneous leishmanaisis is a tropical skin disease of major public health importance (Desjeux, 2004). It is endemic in many tropical and subtropical developing countries.

The World Health Organisation (WHO) considers leishmaniasis to be one of the most serious parasitic diseases and theWorld Health Assembly has advocated a concertation for its control (WHO, 2007). There are about 88 leishmaniasis endemic countries of the world ,of which 72 are developing countries. Absent only from Australia and Antarctica due to the lack of suitable vectors (Cruz et al., 2006). Globally an estimated 12-15 million people are affected and approximately more than 350 million people are at risk of infection . Reports from about 65 countries of the world indicated (Desjeux, 2004).

VL is the most severe form of leishmaniasis, almost always fatal if untreated. Over 90% of the estimated annual incidence of 500,000 VL cases worldwide (Desjeux, 2004). And globally an estimated of 1 to 1.5 million new cases of CL are reported annually (Desjeux, 2004). It is endemic in many tropical and subtropical developing countries protozoan parasites are amongst themost common infectious agents and have serious consequences for socio- economic development (Alvar et al., 2006). CL is endemic in more than 70 countries worldwide, and 90% of cases occur in Afghanistan, Algeria, Brazil, Pakistan, Peru, Saudi Arabia, and Syria (Desjeux, 2004). Surveillance data indicate that the global number of cases has increased during the past decade, as documented in Afghanistan, Bolivia, Brazil, Colombia, Syria and Peru (Davies et al., 2000a; Reithinger et al., 2003 and King et al., 2004).

Increases can be explained in part by improved diagnosis and case notification, (Yadon et al., 2001) but are also a result of inadequate vector or reservoir control, increased detection of CL associated with opportunistic infections (eg, HIV/AIDS), (Molina et al., 2003). Currently, the disease appears to be underestimated and on the rise in several countries. Old World CL is also increasingly seen in immigrants, military personnel, humanitarian aid workers, tourists and travellers from endemic areas. However, imported CL is still missed by most Western physicians (González et al., 2008). Suspected skin lesions need to be analysed with biopsies and tissue smears in order to make an accurate diagnosis (Reithinger et al., 2007).

Leishmanial infection is transmitted to humans through the bite of female hematophagus sandflies of the Phlebotomus genus in the Old World and of the Lutzomyia genus in the New World (Santos et al., 2008). Infection may be also acquired through the blood-borne route (sharing of syringes, blood transfusion), transplacentally, or through solid organ transplantation (Antinori et al., 2008; Dujardin et al., 2008; Oliveira et al., 2008; Riera et al., 2008 and Santos et al., 2008). There are over 20 species of Leishmania that have been recorded as causing human infection which are either zoonotic, or have recent zoonotic origins. The distribution of each species is determined by distribution of its vector, reservoir host, or both (Ashford , 2000 and Desjeux, 2004). Depending on virulence factors of Leishmania species and host immune responses, infection can be manifested as VL, MCL and CL form.

CL is the most common clinical type and is still an important public health problem in the 21st century due to not only environmental risk factors such as malnutrition, genetic disposition, and poverty. the disease still requires improved control tools and the WHO \TDR financed research for leishmaniasis has been more and more focusing on the development of new tools such as diagnostic test, drugs and vaccines. The epidemiology and clinical features of the disease are highly variable due to the interplay numerous factors in the parasites, vectors, hosts and environments involved (Desjeux, 2004).

2.1.7. Clinical symptoms of leishmaniasis

Several Leishmania spp can cause CL in human beings, although most infections probably remain symptomless (Murray et al., 2005). CL is usually noted on exposed parts of the body, mainly arms, face, and legs. The clinical manifestations are extremely diverse including unusual sites and atypical morphologies. Typically, the natural course of the lesions seen in CL. The lesions typically are not painful, but are associated with significant stigma associated with the disease. Women and children are particularly affected. As noted, however, it is increasingly seen in various unusual forms, for example, as fissures on lips, with lupoid features on face and/or psoriasiform plaques on nose (Rahman et al., 2009 and UI Bari and Ejaz, 2009).

Some lesions are characterized by development of nodules, which progress to ulcerative lesions, lasting from between 3 months to three years. Epidermal changes reflect the immune response to the infection, resulting in hyperplasia and epidermal thickening. Within the dermis the collagen matrix is disrupted and fibroblasts are eventually recruited during the healing process (Mehregan et al., 1999). Epidermal disruption results in discharge and eventually dries to form an encrusted ulcer, with a central depression and raised border (Hepburn, 2000). It is in this latter region where parasites are present in dermal macrophages. Resolution usually occurs following the generation of the appropriate Th1 response and the resulting cytokines (IFNγ, TNFα, IL-12) that confer resistance to infection with leukocyte migration resulting in necrosis and formation of a healing granuloma (Ghersetich et al., 1999).

On one end of the spectrum of CL is the classical oriental sore in which spontaneous cure and immunity to reinfection is the result of an effective parasiticidal mechanism. On the other end of the spectrumis diffuse CL in which metastatic cutaneous lesions develop and the patient rarely, if at all, spontaneously develops immunity to the parasite‖ (Ul Bari et al., 2009a).

CL has been also categorized into four different clinical forms: Localized cutaneous leishmaniasis (LCL)

In the localized form the parasite is confined to the skin. After an incubation period of 1 to 12 weeks a papule or bump develops at the site of the insect bite. The papule grows and turns into an ulcer. A typical lesion of the localized form of CL is a painless papule or ulcer covered with an adherent crust of dried exudate. Most people with CL have 1 or 2 lesions varying in size from 0.5 to 3 cm in diameter, usually on exposed parts of the body such as the face, arms or legs. There is, however, considerable variation people may have as many as 200 simple skin lesions some lesions grow but do not ulcerate (nodules) and some Leishmania species also infect the lymphatic system producing lesions along the lymphatic channels (nodular lymphangitis). Secondary bacterial infection is common, causing pain and serious disability. Most lesions heal spontaneously over months or years, leaving permanent scarring with skin thinning. Scarring of leishmaniasis is typical with a depigmented centre and a pigmented border (Reithinger et al., 2007).

LCL lesions vary in severity (eg, lesion size), clinical appearance (eg, classic LCL, vs disseminated leishmaniasis (Turetz et al., 2002), are common and there is a variable tendency for lesions to self-cure within approximately 2– 6 months L. major, (Alrajhi et al., 2002; Momeni et al., 2003 and Nassiri- Kashani et al., 2005). 3–9 months L. mexicana, (Soto et al., 2004), or 6–15 months L. tropica (Dowlati, 1996), L. braziliensis, (Soto et al., 2004), L. panamensis (Soto et al., 2004).

Recidivans

This form appears in around 5% of the participants suffering CL by L. tropica (Alrajhi, 2003) or L. amazonensis (Choi and Lerner, 2001) and is characterised by microsatellite and confluent lesions that relapse and finally ulcerate in the border of previous scars.

Diffuse Cutaneous Leishmaniasis (DCL)

Diffuse leishmaniasis affects only the skin but with generalized skin lesions. This chronic skin disease is caused by L.aethiopica (Alrajhi, 2003) or L.amazonensis (Ashford‚ 2000) or L.mexicana (Lee and Hasbun, 2003) . Post kala-azar dermal leishmaniasis is a form of DCL and a sequel of VL that may appear in affected individuals up to 20 years after the being partially treated, untreated or in those considered adequately treated (Rathi et al., 2005).

M ucocutaneous cutaneous leishmaniasis (MCL)

MCL also known as espumda (Singh and Sivakumar, 2003) , these the parasite may spread to the mucous membranes, especially those of the nose, mouth and throat, and cause extensive damage and disfiguration. It is seenmainly in South America but it can also be caused by species from Old World countries including L. tropica, L. major and L. infantum (Amato et al., 2007). Mucosal lesion develop within two years in 50% of patients and 90% within 10 years of infection (Salman et al., 1999).

2.1.8. Diagnosis of leishmaniasis

The clinical signs and symptoms of all forms of leishmaniasis are non specific and diffuse. Diagnosis of an active leishmaniasis infection can be tricky. To complicate things further the symptoms caused by the infection are very similar to other diseases such as malaria. The longer the patient spends untreated the more at risk the community becomes as the patient will act as a reservoir for the disease. To make matters worse the medical staff want to be sure of the diagnosis before the start of treatment due to the toxic and expensive drugs they will have to administer for the treatment length of 3 weeks (WHO, 2011). Diagnosis of leishmaniasis can be made using different approaches:

2 .1.8.1. Direct visualization or isolation of the parasite

M icroscopic examination

Microscopic examination is probably the most common diagnostic approach used, because more sophisticated techniques are expensive and rarely available at primary, secondary, and tertiary health-care levels in endemic areas. Culture methods are probably the most informative, allowing species identifi cation and characterisation, but require a wealth of technical expertise, and are time consuming and expensive. The sensitivity of these techniques, however, tends to be low and can be highly variable, depending on parasite number and dispersion in biopsy samples, technical expertise, and culture media. Molecular parasitological diagnosis for cutaneous leishmaniasis was developed extensively during the past decade, and has been recently reviewed (Reithinger et al., 2007).

The confirmatory diagnosis of leishmaniasis relies on either the microscopical demonstration of Leishmania amastigotes in the relevant tissues aspirates or biopsies such as bone marrow, spleen, lymph nodes or liver, skin slit smears or biopsies (Singh et al., 2005) or in the peripheral blood buffy coat (Liarte et al., 2001).

The smears can be also stained with Romnowsky‘s, hemotoxyline eosine (H & E) or immunoperoxidase stains. The amastigotes are readily seen in smears or touch preparations of infected tissue stained with Giemsa‘s stain, preferably at pH 7.2 rather than the pH 6.8 normally used in haematology. To ensure that the visualized structures are amastigotes, rather than other dot like structures (e.g., Histoplasma spp.¸ platelets), an experienced observer should look for the characteristic size (2-4 mm in diameter), shape (round to oval), and internal organelles, the nucleus and kinetoplast. It is important to discern the kinetoplast. With Giemsa staining, the cytoplasm typically takes pale blue and the nucleus and kinetoplast take purple-pink colour (Herwaldt, 1999). The immunoperoxidase stain provides improved sensitivity in cases of cutaneous and mucocutaneous leishmaniasis (Herwaldt, 1999). Various clinical samples can be used depending on the clinical forms of diseases and aimed sensitivity.

Spleen aspirate and biopsy

For VL in the immunocompetent patients, best samples are those obtained from spleen aspirations (Herwaldt, 1999). The splenic aspirate is the best with sensitivity > 94 percent than other tissue aspirations and in experienced hands the iatrogenic splenic bleeding can be minimized. Although many practitioners are reluctant to take spleen aspirates, others have no hesitation, and even use this method to monitor treatment. It is vital to use the correct technique and equipment with confidence, so that the capsule of the spleen is penetrated by a fine needle for only a fraction of a second (Bryceson, 1987). The thinnest needle possible, preferably, 21-gauge (0.8 mm) should be used to minimize the risk of complications such as haemorrhage of the spleen (Chulay and Bryceson, 1983).

However, even in experienced hands, the risk cannot be zeroed and fatal bleeding can occur in 2/10,000 patients, inspite of prior precautions in the form of >40000/ ml platelet count and a good prothrombin time control measures are adopted (Bryceson, 1987). Part of the splenic aspirate can be used to make smears for direct microscopic examination and the rest should be cultured. In splenic aspirate smears the amastigotes of Leishmania appeared ovoid in shape and measure about 3 x 5 μm in size. We have observed that amastigotes which are short and more stout measuring as big as 4.5 x 5 μm are resistant to sodium antimony gluconate (SAG) while patients who show elongated amastigotes in their specimen are sensitive to standard doses of SAG (Singh et al., 2005) and this morphological observation needs confirmation.

The parasites appear purple blue with central nucleus and a rod shaped structure at the right angle of nucleus, both pink in colour. This rod shaped structure is an extrachromosomal DNA mass known as kinetoplast. The splenic apirates can also be used for determining the disease prognosis and therapeutic response by estimating parasite load by counting the number of amastigotes in the smears in relation to the white blood cell counts (Singh et al., 2005). A logarithmic scale from 0 (no parasites in 1,000 microscopic fields) to 6+ (greater than 100 parasites per microscopic field) can be applied (Chulay and Bryceson, 1983).

Liver biopsy

Demonstration of the parasites in the liver aspirates and biopsies is another option. The sensitivity of liver biopsies has been reported by various authors as low as 40 percent, when most of the amastigotes are colonized within the Kuffer cells to as high as 90 percent. Liver aspiration should also be attempted with utmost care as in the case of spleen puncture not to tear the capsule (El Hag et al., 1994).

Bone marrow aspiration

Bone marrow obtained from sternal or iliac crest puncture is a much safer but a painful method. It is less likely to demonstrate parasites in direct stained films (Singh et al., 2005) and in most studies the sensitivity ranges from 76-85 percent. However, on culture it can give positive results in up to 80 percent of the cases (Singh et al., 2005).

Lymphnode fine needle aspiration cytology (FNAC) and biopsy

Lymph gland puncture may give positive results in up to 40-50 percent of the kala-azar cases but its sensitivity has been found much higher (58.6%) in cases of cutaneous leishmaniasis in a study carried out in Brazil (Romero et al., 1999). The aspirate is extracted from any enlarged lymph gland after injecting sterile normal saline and the aspirate is subjected to both direct examination and culture to give the best chance of diagnosis. In CL, the lymph nodes draining from the lesion sites are most yielding. Parasites may be scanty and are mostly extracelullar in slide preparations, so these may have to be examined for at least 15 min using oil immersion before the diagnosis can be confirmed (Singh, 2006).

Blood buffy coat

Rarely the amastigotes can be demonstrated in the buffy coat of peripheral blood. Such a parasitaemia is common in severely immunocompromised patients such as AIDS (Martinez et al., 1993) and patients on immunosuppressive therapy (Maggi et al., 2004). While some authors have found sensitivity up to 53 percent (Delgado et al., 1998) other have found this method very poorly (7.6%) sensitive (Navin et al., 1990).

Tegumentary leishmaniasis

The routine diagnosis of CL patients depends on examination of skin lesions using smears and cultures of dermal scrapings or examination of sections obtained from a skin biopsy. Conventionally 3-5 aspirates from different lesions or portions of lesions are obtained. This is best done by injecting 0.1 ml sterile normal saline into the lesion site so that it inflates a bit. For ulcerative lesions, needle (23-27 gauge) is inserted through intact skin into dermis of active border. Small-gauge needles are appropriate for facial lesions. The needle is repeatedly moved back and forth under skin, tangentially to ulcer, simultaneously rotating the syringe and applying suction, until pink-tinged tissue fluid is noted in hub of needle (Mimori et al., 2002).

The most sensitive method was a combination of thin smears made from superficial scrapings of the ulcers and inoculation of culture medium with either aspirates or scrapings. Ability to cultivate Leishmania was correlated with the concentration of amastigotes seen on thin smears. Leishmania were cultured in 42 (27%) of 153 patients with no amastigotes found in 400 oil-immersion fields and in 174 (83%) of 209 patients with at least 1 amastigote (Mimori et al., 2002). Whatsoever sample collection method is used, each aspirate should be collected into separate tubes of Novy-MacNeal-Nicolle (NNN) culture medium to make separate slides for microscopy. If punch-biopsy samples are intended, one to two full thickness of skin at active border of lesion including some non ulcerated tissue should be obtained and used for culture and histopathology. For dermal scrapping, 3-5 dermal scrapings from different lesions or portions of lesions should be taken (Singh, 2006).

2 .1.8.2. Culture examination

Isolation of the causative agent is most specific diagnostic criterion and also to characterize the organisms up to species or genotype level. The promastigote form can be culture isolated from these specimens on solid NNN medium having 20-30 percent rabbit blood or liquid Schneider‘s insect medium. Up to 90 percent of the active kala azar cases will grow promastigotes in their splenic and liver aspirates (Manson-Bahr, 1987). Various other liquid media such as M199, Tobies medium supplemented with foetal calf serum can be used. Human urine has been successfully used in place of foetal calf serum in in vitro culture of L. donovani (Singh et al., 2000).

The promastigotes in vitro transformation in the NNN medium usually starts after 3 days of incubation at 22-26 0C in a BOD incubator. The wet mounts prepared from the liquid part of this diphasic medium will show several motile organisms, the details of which can be delineated after staining the smear with Giemsa or any other Romnowsky‘s stain or by using fluorescent antibody staining (Singh et al., 2005). Culture based diagnosis of MCL has low sensitivity as the organisms are often scant. The biggest handicap is culture contamination at early stages, even in best laboratory setups (Singh et al., 2005).

2 .1.8.3. Isolation and inoculation in experimental animals

Alternative methods to isolate the parasite can be used. Inoculation of the clinical material obtained either into a susceptible BALB/c mouse or into a hamster footpad or nose may improve the yield. Histopathologic evaluation of biopsy samples of animal lesions may be characteristic but is rarely specific enough to make a diagnosis without identification of the amastigote. From Columbia, compared seven methods of diagnosing leishmaniasis in 177 patients presenting with lesions of the skin or mucosa. Microscopic methods of visualizing amastigotes in tissue samples were less sensitive than the Leishmania isolation methods. The aspirate culture and biopsy-hamster methods employed in this study proved most sensitive of the four methods for the recovery of parasites. All methods were less sensitive in lesions of greater than 6 months duration than in lesions of more recent onset. Mucosal lesions were best diagnosed by the culture or hamster inoculation of a macerated mucosal biopsy (Singh, 2006).

The diagnosis by inoculation of hamsters was achieved within 2 to 12 wk, a mean of 34.5 days (Weigle et al., 1987). In another study from Kenya (Shatry et al., 1988) on VL, portions of splenic or subcutaneous saline aspirates from suspected visceral or CL patients were inoculated into NNN medium with an overlay of Schneider‘s medium or Schneider‘s medium alone for routine parasitological diagnosis. The remaining portions of the aspirates were used for preparing Giemsa-stained smears and for subcutaneous inoculation into hind foot-pads of BALB/c mice. Saline aspirates obtained from the foot-pads 2-14 days after inoculation were inoculated into Schneider‘s medium and examined for promastigotes. Parasite isolation was achieved from 90 percent of confirmed leishmaniasis patients by culture method alone. Mouse foot pad aspiration demonstrated parasites in 95 percent of all patients, and in over 80 percent of the confirmed cases of Leishmaniasis (Singh, 2006).

Combined culturing and aspirate smear examination was more efficient than foot pad inoculation alone for the demonstration of leishmanial infection. Foot pad aspiration does not entail killing animals and was sensitive for parasite isolation, it may be a useful short term adjunct to existing parasite isolation methods, especially under field conditions where the risks of culture contamination may be high (Shatry et al., 1988).

2 .1.8.4. Immunological methods of diagnosis of CL

The hallmark of VL is hyperimmunoglobulinaemia, while in case of CL and MCL, the humoral immune response is extremely poor. Exploiting this host parasite interaction, for the diagnosis of VL a number of antibody detection methods have been developed from time to time. Some of these tests include indirect haemagglutination (IHA), counter current immune-electrophoresis (CCIEP), immunodiffusion (ID) and several others (Boelaert et al., 2004 and Singh et al., 2005) . These tests are cumbersome and lack sensitivity and specificity and hence not commonly used but the interested readers can find more information from some recent reviews (Singh et al., 2005).

Fluorescent antibody test

The indirect fluorescent antibody (IFA) test is one of the commonly used tests for anti-leishmanial antibody detection using fixed promastigotes. 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. Titres above 1:20 are significant and above 1:128 are diagnostic. However, there is a possibility of a crossreaction with trypanosomal sera (Singh et al., 2005). The sensitivity of these tests varies extremely from as low as 28.493 to 86.6 percent (Iqbal et al., 2002). This can be overcome by using Leishmania amastigotes as the antigen instead of the promastigotes. To detect the antigen (amastigotes) in the tissue sections or smears, fluorescent dye conjugated antibodies can be used as tracers. This test is known as direct fluorescent test. The direct fluorescence test is more useful in the diagnosis of CL, MCL and PKDL. In place of fluorescence, horse radish peroxidase (HRP) can be used to tag the antibody. This will not require fluorescence microscope and the stained slides can be stored for long time (Singh, 2006).

Direct agglutination test

The direct agglutination test (DAT) is a highly specific and sensitive test. It is cheap and simple to perform making it ideal for both field and laboratory use. DAT in various studies has been found to be 91-100 percent sensitive and 72- 100 percent specific (Tavares et al., 2003). The test can be carried out on plasma and serum. For long time DAT remained first line diagnostic tool in resource poor countries. The method uses whole, stained promastigotes either as a suspension or in a freezedried form. The freeze-dried form is heat stable and facilitates the use of DAT in the field (Abdallah et al., 2004). However, the major disadvantage of DAT is the long incubation time of 18 hour and the need for serial dilutions of blood or serum. Also the DAT has no prognostic value for evaluating the parasitological cure of the disease, as the test may remain positive for several years after cure.

Developed a fast agglutination-screening test (FAST) for the rapid detection (<3 h) 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. The FAST offers advantages over the DAT as it uses freeze dried antigen, which gives more antigen stability, reproducibility, specificity and sensitivity (Schoone et al., 2001).

Enzyme linked immunosorbent assay (ELISA)

ELISA is a valuable tool and one of the most sensitive tests for the serodiagnosis of visceral leishmaniasis. The test is useful for laboratory analysis or field applications and to screen a large number of samples at a rapid pace. With the advances in automation, ELISA can be performed easily and is adaptable for use with various antigens such as whole cytoplasmic (soluble antigen, SA), purified antigens such as fucosemanose (Palatnik-de-Sousa et al., 1995) defined, synthetic peptides and recombinant proteins as antigen (Maalej et al., 2003). The sensitivity and specificity of ELISA is greatly influenced by the antigen used. Beside the most commonly used soluble promastigote antigen, several antigenic molecules have been reported and their negative and positive predictive values (NPV & PPV) compared (Maalej et al., 2003).

A recombinant antigen, rK39 has been shown to be specific for antibodies arising during VL caused by members of the L. donovani complex. It is highly sensitive and predictive for onset of acute disease and evokes high antibody titres in VL patients (Singh et al., 2002). In addition, rK39 ELISA, has a high predictive value for detecting VL in immunocompromised persons, like AIDS patients (Singh et al,. 2005). This antigen is now commercially available in the form of antigen-impregnated nitrocellulose paper strips adapted for use under field conditions. However, reports from Sudan and other countries revealed that this antigen showed decreased sensitivity and specificity. In Sudan the rK39 ELISA test is reported to miss 7 percent parasitologically proven cases (Zijlstra et al., 2001). In its strip test format the sensitivity is further compromised to only 67 percent in Sudan, 71.4 percent in Southern Europe and 60 to 90 percent in Brazil (Schallig and Oskam, 2002 and Carvalho et al., 2003).

Besides rK39, two more recombinant proteins (rK26 and rK9) have been cloned from L. chagasi kinesin gene (Bhatia et al., 1999). A significant difference between K9 and K26 is the presence of 11 copies of a 14 amino acid repeats in the open reading frame of K26. The region flanking the repeats of K26 shares a 69 percent identity with the open reading frame of K9. Therefore, a need was felt to clone the kinesin antigen from any Old World species of Leishmania.Very recently, we have cloned and characterized a recombinant antigen from an Indian isolate of L. donovani strain KE16. The antigen (rKE16) is found to be 100 percent sensitive and specific. In fact it has better sensitivity than rK39 which showed 98 percent sensitivity for the diagnosis of Indian kala- azar and PKDL (Sivakumar et al., 2006). It also showed 100 percent concordance with rK39 in sera from leishmaniasis patients from China, Pakistan, and Turkey109. This antigen has now been commercialized and has got tremendous potential for the serological diagnosis of VL worldwide.

Immunoblotting

Serodiagnosis using immunoblotting of soluble antigens has been attempted and reported highly sensitive and specific. The band pattern can correlate with disease stages (Ravindran et al., 2004).

Using cytoplasmic, soluble antigens from 5 Indian strains of L. donovani and three L. major strains from Pakistan separated by SDS-PAGE and electrotransferred on nylon membrane followed by Western blotting with Indian PKDL patients, a ~72-74 kDA antigen band was found to be most predominant (Singh et al., 2005). The commercially available electrochemiluminis cent kit (ECL, Amersham, UK) enhances its sensitivity, several-folds. It also has an added advantage of permanent documentation.

Rapid antibody detection methods

In most of the Leishmania endemic areas resources are limited in terms of poor or non-availability of electricity, poor laboratory set up and lack of equipment. Therefore, need of rapid, simple and easy to perform tests has always been felt. With this objective two rapid tests have been developed, one by InBios (USA) which uses Lc-rK39 antigen and the other one is by Sa pn Diagnostic Limited (India) which uses Ld-rKE-16 antigen. Both are commercially available and are based on membrane filtration technology (Singh, 2006).

Antigen detection

Antigen detection test would, in principle provide better means of diagnosis of active leishmaniasis. Since antigen levels are expected to theoretically correlate with the parasite load, the antigen detection may be an ideal test in immunocompromised patients, where antibody response is very poor. The detection of antigen in the patient‘s serum is complicated by the presence of high level of antibodies, circulating immunecomplexes, serum amyloid, rheumatoid factor and autoantibodies, all of which may mask immunologically important antigenic determinants or competitively inhibit the binding of free antigen. Recently, a latex agglutination test (KATEX) for the detection of leishmanial antigens in the VL patients‘ urine has been developed (Attar et al., 2001). The results obtained with KATEX using samples collected from different foci of VL indicated that the test worked well regardless of the geographical origin of samples. The test had 100 percent specificity and sensitivity between 68-100 percent. Whether the test has applications for the detection of asymptomatic cases of VL and monitoring therapy is yet to be confirmed.

Diagnosis using amastigote specific antigen

The in vivo parasitic stage of Leishmania in humans is amastigote form and it is not difficult to appreciate that antigens specific or prepared from this stage of L. donovani, would be more ideal. However, due to difficulty in maintaining the culture of the amastigote stages in bulk quantity not many studies are available in the literature. Otherwise also the sensitivity and specificity of crude antigens prepared from amastigotes have not been found superior to the recombinant antigen rK39 (Sreenivas et al., 2002 ).

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. No cross-reaction occurs with Chagas‘ disease, but some cross reactions are found with cases of glandular tuberculosis and lepromatous leprosy. LST is used as an indicator of the prevalence of cutaneous and mucocutaneous leishmaniasis in human and animal populations and successful cure of the visceral leishmaniasis. During active kala-azar, there will be no or negligible cell mediated immune response (Liarte et al., 2001).

However, the Leishmanin antigen is not commercially available and no field study has been carried out in Indian subcontinent. Despite the availability of large number of serological tests, no serological method is helpful for cutaneous and mucocutaneous leishmaniasis because antibodies tend to be undetectable or present in low titre due to poor humoral response (Amaral et al., 2000 and Ahluwalia et al., 2004). One of the big drawbacks of this approach is the lack of standardized leishmanin (Bern et al., 2006). The Leishmanin Montenegro skin test is simple to use and highly specific but produces false negative results in some affected patients and it does not distinguish between previous and present infections (Escobar, 1992).

2 .1.8.5. Molecular methods

Molecular biology is increasingly becoming relevant to the diagnosis and control of infectious diseases. Information on DNA sequences has been extensively exploited for the development of polymerase chain reaction-based assays for various applications in the understanding of the parasite and the diseases. With the advent of nucleic acid engineering and recombinant technology, a number of strategies have been developed to produce recombinant proteins for diagnostic purposes (Tavares et al., 2003 and Singh et al., 2005).

A variety of nucleic acid detection methods targeting DNA and RNA genes have been developed. However, amongst all the molecular advances gene amplification techniques have been most rewarding as far as diagnosis and disease management is concerned.

Polymerase chain reaction (PCR)

Amongst the molecular methods used for clinical diagnosis, PCR has been proved to be most sensitive and specific technique, albeit limited to tertiary