Recent advances in Leishmaniasis

Dr.Pinaki Chattopadhayay

17/02/2011

INTRODUCTION

• Leishmania is a blood and tissue flagellates

• Disease caused by this parasite is called Leishmaniasis

• Of two types : Old world Leishmaniasis and New world Leishmaniasis

• Old world leiashmaniasis : Cutaneous and Visceral

• New world leishmaniasis : Cutaneous , Mucocutaneous and Visceral

Taxonomy

• Domain: Eukaryota

• Phylum: Euglenozoa

• Class: Kinetoplastida

• Order: Trypanosomatida

• Genus: Leishmania

• Species: Leishmania species (more than 20)

Species, disease and their geographical distribution

Old world Leishmaniasis

Form of disease Geographical distribution

L. donovani Visceral leishmaniasis,Kala-azar, PKDL

Indian subcontinenet, Africa, Middle east ,Kenya

L.infantum VL, China ,Mediterranian coast , Middle East, Africa ,China

L.archibaldi VL, old world cutaneous leishmaniasis

Ethiopia, Sudan, KenyaEast Africa , Sudan

L. tropica Old world cutaneous leishmaniasis, Oriental sore

Central Asia, Middle east ,Africa, Indian subcontinent

L.major Old world cutaneous leishmaniasis

Indian subcontinent, Central Asia, Africa,Middle east

L.aethiopica Old world cutaneous leishmaniasis, diffuse cutaneous leishmaniasis

Ethiopia, Kenya

New world Leishmaniasis Form of disease Geographical distribution

L.braziliensis complex L. braziliensis, L. panamensis L. guyanensis L.peruviana

Mucosal leishmaniasis(Espundia) Mucosal leishmaniasisCutaneous leishmaniasisCutaneous leishmaniasis (Uta)

South America,BrazilPanamaGuyanaPeru, Western Andes

L. mexicana complex L. mexicana, L. venezuelensis L. amazonensis

Cutaneous leishmaniasisDiffuse cutaneous leish maniasis Cutaneous leishmaniasis,Diffuse cutaneous leishmaniasisCutaneous leishmaniasis,Diffuse cutaneous leishmaniasis

Central America , Mexico,

Venezuela

Amazonian basin

L.chagasi American visceral leishmaniasis Latin America

HISTORY

• Descriptions of conspicuous lesions similar to cutaneous leishmaniasis (CL) has been discovered on tablets from King Ashurbanipal from the 7th century BC, some of which may have been derived from even earlier texts from 1500 to 2500 BC.

• Muslim physicians including Avicenna in the 10th century AD gave detailed descriptions of what was called Balkh sore.

• In 1756, Alexander Russell, after examining a Turkish patient, gave one of the most detailed clinical descriptions of the disease.

• Physicians in the Indian subcontinent would describe it as Kala-azar (pronounced kālā āzār, the Urdu, Hindi and Hindustani phrase for black fever, kālā meaning black and āzār meaning fever or disease).

• As for the new world, evidence of the cutaneous form of the disease was found in Ecuador and Peru in pre-Inca potteries depicting skin lesions and deformed faces dating back to the first century AD.

History contd…

• 15th and 16th century texts from the Inca period and from Spanish colonials mention "valley sickness", "Andean sickness", or "white leprosy", which are likely to be CL.

• Who first discovered the organism is somewhat disputed. It is possible that Surgeon major Cunningham of the British Indian army saw it first in 1885 without being able to relate it to the disease.

• Peter Borovsky, a Russian military surgeon working in Tashkent, conducted research into the etiology of oriental sore, locally known as Sart sore, and in 1898 published the first accurate description of the causative agent, correctly described the parasite's relation to host tissues and correctly referred it to Protozoa.

History contd…. In 1901, Leishman identified certain organisms in smears taken from the spleen of a

patient who had died from "dum-dum fever" (Dum Dum is an area close to Calcutta) and proposed them to be trypanosomes, found for the first time in India.

A few months later Captain Charles Donovan (1863–1951) confirmed the finding of what became known as Leishman-Donovan bodies in smears taken from patients in Madras, India.

But it was Ronald Ross who proposed that Leishman-Donovan bodies were the intracellular stages of a new parasite, which he named Leishmania donovani.

The link with the disease kala-azar was first suggested by Charles Donovan, but was conclusively demonstrated by Charles Bentley's discovery of Leishmania donovani in patients with kala-azar.

The disease was a major problem for Allied troops fighting in Sicily during the Second World War, and it was then that research by Leonard Goodwin showed that pentostam was an effective treatment.

Classification • The ideal classification for an asexual organism at the level of species combines the

maximum biological homogeneity within each species , but separates biologically different organism .

• Among the techniques , isoenzyme analysis have been found to describe strains at a level that allows the construction of phenograms (taxonomic relationships among organisms) and cladograms (ancestral relations between organisms).

• Using a panel of c.12 enzymes ,it is possible to identify and distinguish most, but not all of the currently accepted species .

• The group of strains that share a given pattern of enzymes electrophoretic mobilities are known as “zymodeme”. A given species may contain a number of zymodemes indicating subspecies.

• The main disadvantage of isoenzyme analysis is the lack of standardisation between laboratories

Classification

• Of the other techniques , the resolution of of restriction fragment length polymorphism (RFLP) and karyotype analysis has been found to be too fine except for studies at the population genetic level .

• Microsatellite DNA analysis is the latest technique being used with Leishmania and seems well suited to resolve taxonomic issues .DNA and ribosomal RNA sequence information hold much promise and have given valuable result s at the generic level .

• Monoclonal antibody and DNA probes , with or with out PCR amplification , are of value in identification of stocks that have been well classified .

Vector and the Host

• Leishmania -two host

• Invertebrate, i.e the sandflies, develops in gut and tranmission via mouth

• Vertebrate hosts i.e mammals( man and also dog in some areas ) in which parasite resides within the phagolysosomal system of mononuclear phagocytic cells ,typically macrophages.

• It is mainly a zoonosis , man is the incidental host , sometimes human –vector –human transmission also occur in some part of the world .

• The invertebrate host hosts are all phlebotomine sandflies of two genera ;Phlebotomus( P.argentipes, P.orientalis, P.martini) in the Old world and Lutzomiya(L.longipalpis, L. vetulus ) and Psychodopygus in the New world.

• These are small ,hairy ,dipteran flies of the family Psychodidae , in which only the females feed on blood and transmit disease

Phlebotomus Lutzomiya

Different forms of the parasite

• Amastigote:• it is found in man and other mammalian hosts .• Found inside the monocyte , polymorpho nuclear leucocytes or endothelial cells • Small, round to oval bodies measuring 2-3µm in length • Also known as LD bodies • Stained well with Giemsa or Wright

Amastigotes of Leishmania donovani are seen in an impression smear. The nuclei of L. donovaniis are clearly visible (Giemsa stain, 1000x).

Promastigote

• It is found in the digestive tract of sand fly (vector) and in the culture media

• Long , slender and spindle shaped • 15-25 µm in length and 1.5-3.5 µm in breadth • Single nucleus situated in the centre • The kinetoplast lies transversely near the anterior part , flagellum is single • With leishman stain , cytoplasm appears blue , the nucleus pink, ,

kinetoplast bright red

•Promastigote in culture, Giemsa stained•Elongated shaped, with free flagellum at the anterior end•No undulating membrane•A conspicuous rod-shaped kinetoplast

Morphological forms

They possess a single nucleus,A single kinetoplast consists of parabasal body and an adjacent dot like blepharoplast ( connected with delicate fibrils) and Singe flagellum .The portion of the flagellum extending from the blepharoplast to the surface of the body of the parasite is known as axoneme

LIFE CYCLE

Types of presentation

• Visceral leishmaniasis (VL), also known as kala azar, is the most severe form of the disease, which, if untreated, has a mortality rate of almost 100%. It is characterized by irregular bouts of fever, substantial weight loss, swelling of the spleen and liver, and anaemia.

• Mucocutaneous leishmaniasis (MCL), or espundia, produces lesions which can lead to extensive and disfiguring destruction of mucous membranes of the nose, mouth and throat cavities.

• Cutaneous leishmaniasis (CL) can produce large numbers of skin ulcers—as many as 200 in some cases—on the exposed parts of the body, such as the face, arms and legs, causing serious disability and leaving the patient permanently scarred.

• Diffuse cutaneous leishmaniasis (DCL) never heals spontaneously and tends to relapse after treatment. The cutaneous forms of leishmaniasis are the most common and represent 50-75% of all new cases.

Pathogenesis of VL

• Sand fly liberates biologically active substances , which promote infectivity of promastigotes by partially deactivating fixed macrophages in the skin.

• • The promastigote activate complement by alternate pathway producing activated products

of complement such as C3b or C3bi.

• These activated products bind with two specific receptors present on the outer membrane of promastigotes (63 kDa mw glycoprotein and lipophosphoglycan) . These receptors bind with complement receptors present on the surface of macrophages either directly or through bound C3b or C3bi receptors .

• Promastigotes phagocytosed by macrophages are transformed into amastigotes and multiply by binary fission within phagolysosomes of the macrophages .

• Amastigotes subsequently invade throughout the reticuloendothelial system of the spleen, liver , bone marorw , and lymph nodes and multiply in large numbers .

• Increased number of macrophages produce progressive hypertrophy of the organs, replace lymphoid follicles in the spleen and also haemopoetic tissue in the bone marrow .

• Proliferation and destruction of RE cells of the internal organs and heavy parasitisation of external organs by parasitised cells are the characteristic pathological changes in VL

Visceral leishmaniasis

Leishmania virus

• It is a double stranded RNA virus

• Persistently infects some strains of Leishmania

• Possibility that the presence of this virus may be able to alter the parasite phenotype and may affect disease pathogenesis.

• The virus has been detected in cultured parasites and also been detected in human biopsy samples prior to manipulation in culture

Host immune response

• Leishmaniasis is a disease in which the clinical diversity reflects a complex interplay between the virulence of the infecting species and the host's immune response.

• At one extreme, localized cutaneous disease demonstrates a vigorous immune response, with most cases resolving without intervention.

• This form of disease exhibits a helper T-cell subtype 1 (TH1) immune response, with interleukin 2, interferon g, and interleukin 12 as the prominent cytokines that induce disease resolution.

• At the other extreme, with visceral or diffuse cutaneous disease, patients exhibit relative anergy to the Leishmania organism and have a prominent helper T-cell subtype 2(TH2) cytokine profile.

Leishmaniasis and The Immune System

• In order to develop a successful parasitic relationship with its host, the leishmania must evade both the innate and adaptive immune responses.

• When leishmania first enters the human body, it is in the promastigote form. Promastigotes are engulfed by macrophages but are resistant to proteolysis and degradation in the phagosome.

• Once inside the macrophage, the organism is termed an amastigote. By continuing to live inside the macrophage, leishmania effectively avoids the humoral branch of the immune system.

• During each of the steps described, the protozoa evade and at times manipulate the human immune system and avoid digestion.

Leishmaniasis and The Immune System

• After being engulfed, the leishmania must endure harsh conditions inside the phagosome. For example, the macrophage uses an oxidative burst to destroy foreign material in the phagosome. This process consists of an attack by superoxide and hydroxyl radicals on the parasite.

• Leishmania produces acid phosphatases on its surface which inhibit this burst.

• In addition to the oxidative burst, macrophages often attempt to degrade parasites with acidic enzymes. This occurs when lysosomes fuse with the phagosome. The protozoa is resistant to this attack as well because it has a proton pump in the surface which allows its intracellular pH to remain close to neutral.

• Also, the protozoan molecule lipophosphoglycan (LPG) plays an active role by inhibiting lysosomal enzymes.

Recent studies

Cellular and Humoral Immunity in Leishmaniasis

• Because Leishmania manipulates the complement system in order to expedite phagocytosis, humoral immunity is largely ineffective against the organism.

• This has been proven in many experiments. For example, the course of the infection is unaltered in animals that have been depleted of B-cells.

• Thus, the current reasoning is that cell mediated immunity is critical in producing immunity to Leishmania.

• This idea was further explored in Reiner and Locksley's seminal experiments in the 80's.

• In a key experiment, they showed that athymic mice developed Leishmaniasis whereas control mice did not. Thus, Reiner and Locksley proved that immunity to the disease was regulated by T cells.

Recent studies

• A further set of experiments were key not only in furthering knowledge about the disease but also resulted in the development of the TH1/TH2 paradigm of immunity, showed that various strains of mice differed in their susceptibility to the disease.

• For example, C57/BL6 mice recovered after a L. major challenge, whereas Balb/C mice did not.

• Cytokine studies showed that the serum from the resistant C57/BL6 mice contained high levels of Interferon Gamma and IL-2, but low levels of IL-4 and IL-10.

• Conversely, the susceptible BALB/C mice had low levels of Interferon Gamma and IL-2, but high levels of IL-4 and IL-10. (Locksley, 1991)

Recent studies

• In further studies it was clearly demonstrated that these cytokines are responsible for biasing the nature of the immune response.

• It was found that treating a BALB/C susceptible mouse with anti-IL-4 after infection with Leishmaniasis results in recovery. Therefore, inhibiting IL-4 leads to clearing of the pathogen.

• Resistant C57/BL6 mice were treated with anti-Interferon Gamma after infection with Leishmaniasis and then died.

• Therefore, Interferon Gamma is essential in eliminating Leishmania. These studies suggested that IL-2 and Interferon Gamma are key cytokines in directing an immune response toward intracellular pathogens.

• Conversely, IL-4 and IL-10 gear the immune response toward the clearance of extracellular pathogens.

• Further experiments with this model led to further developments of the TH1/TH2 paradigm. (Reiner, 1995)

Signs and symptoms

• Delayed hypersensitivity reaction as determined by leishmanian skin test and invitro lymphocyte response to leishmanial antigen is completely absent during infection .

• The delayed hypersensitivity , however develops again after successful treatment .

• The intact CMI confers protection agaist the infection .

• Profound hyperglobulinemia-production of large volume of polyclonal non-specific immunoglobulins especially IgG and also specific antileishmanial antibody .

• The circulating antibodies are not protective

Signs and symptoms

• Persons who have recovered from kala-azar are immune from re-infection

• Anorexia ,wasting seen in the disease possibly mediated by cytokines such as TNF and Interleukines.

• Onset of disease is gradual or sudden ( incubation period 3-6 months )

• Presents with fever, hepatosplenomegaly , haematological abnormalities etc.

• Dry skin , dull hairs, skin on hands, mouth ,fore-head becomes grayish and dark coloured ( Kala-azar, India)

Leishmania/HIV Co-infection

• Leishmania/HIV co-infection is emerging as an extremely serious, new disease and it is increasing.

• Leishmania/HIV co-infections are considered a real threat, especially in south-western Europe where between 25% and 70% of adult VL cases are related to HIV and where 1.5%to 9% of AIDS cases suffer form newly acquired or reactivated VL.

• Intravenous drug users have been identified as the main population at risk.

• It is anticipated that the number of Leishmania/HIV co-infections will continue to rise in the coming years and there are indications that cases are no longer restricted to endemic areas.

• The overlapping geographical distribution of VL and AIDS is increasing due to two main factors: the spread of the AIDS pandemic in suburban and rural areas of the world, and the simultaneous spread of VL from rural to suburban areas.

• There are important clinical, diagnostic, chemotherapeutic, epidemiological and economic implications of this trend.

Leishmania/HIV Co-infection

• Although people are often bitten by sandflies infected with Leishmania protozoa, most do not develop the disease.

• However, among persons who are immunosuppressed (e.g. as a result of advanced HIV infections, immunosuppressive treatment for organ transplants, haematological malignancy, auto-immune diseases), cases quickly evolve to a full clinical presentation of severe leishmaniasis.

• AIDS and VL are locked in a vicious circle of mutual reinforcement.

• On the one hand, VL quickly accelerates the onset of AIDS (with opportunistic diseases such as tuberculosis or pneumonia) and shortens the life expectancy of HIV-infected people.

• On the other hand, HIV spurs the spread of VL. AIDS increases the risk of VL by 100-1000 times in endemic areas.

Leishmania/HIV Co-infection

• This duo of diseases produces cumulative deficiency of the immune response since Leishmania parasites and HIV destroy the same cells, exponentially increasing disease severity and consequences.

• VL is considered a major contributor to a fatal outcome in co-infected patients. Lately, however, use of tritherapy, where it is available, has improved the prognosis for Leishmania/HIV cases.

• Leishmaniasis can be transmitted directly person to person through the sharing of needles, as is often the case among intravenous drug users.

• This group is the main population at risk for co-infection.

• Based on a study done in Spain, Leishmania spp were detected by PCR technique, in 65 (52%) of 125 syringes collected in southern Madrid, in 1998, and in 52 (34%) of 154 collected in southwestern Madrid in 2000-01.

Leishmania/HIV Co-infection

• Specific Problems • Leishmania/HIV co-infections impose specific difficulties in terms of diagnosis and

treatment. The usual clinical features (fever, weight loss, liver and spleen enlargement, inflammation of the lymph nodes) are not always present.

• The clinical diagnosis can also be made difficult by associated diseases such as cryptosporidium, disseminated cryptococcosis, cytomegalovirus infection or mycobacterial infection.

• The serological diagnosis is falsely negative in 42.6% of co-infected patients . HIV-positive patients have difficulty in producing antibodies against new infectious agents, especially at a late stage or during relapses.

• Consequently, there is a need to use two or more serological tests and antigens freshly prepared in the laboratory to increase sensitivity.

• Although multiple localizations are frequent (blood, skin, digestive tract, lungs, central

nervous system), parasitological diagnosis can be difficult and has to be repeated to orient the treatment.

• Bone marrow aspirate (BMA) remains the safest and most sensitive technique, but spleen aspirate and liver biopsy are also used. When BMA cannot be performed, the search for Leishmania can be conducted in peripheral blood samples.

Leishmania/HIV Co-infection • Treatment for co-infected patients is aimed at clinical and parasitological cures and prevention of relapses.

Unfortunately, in such patients treatment failure, relapses due to drug resistance and drug toxicity are very common.

• In south-western Europe, follow-up studies using pentavalent antimonials, the same first-line drug used to treat classic leishmaniasis, show a positive response in 83% of cases. However, 52% of patients relapse within a period of one month to three years, with the number of relapses ranging from one to four.

• The main alternative drugs include pentamidine, amphotericin B and amphotericin B encapsulated in liposomes. This encapsulation reduces the occurrence of side-effects, but relapses still occur and the drug remains extremely expensive.

Epidemiological Changes : • Leishmania/HIV co-infections can lead to epidemiological changes which modify the traditional patterns of

zoonotic VL. Co-infected patients harbour a high number of Leishmania in their blood so there is also a risk of them becoming reservoirs of the disease (that is, infective for the sandfly vector) as in anthroponotic foci in Bangladesh, India, Nepal and East Africa. Consequently, there is an increased risk of future epidemics.

• Experimentally, sandflies can be infected through a blood meal containing a very small quantity of blood from co-infected patients. The quantity may be less than the content of a needle. As 71.1% of co-infected patients in south-western Europe are intravenous drug users, transmission of Leishmania has occurred through the sharing of syringes in this population group.

PKDL

• It is a non-ulcerative lesion of the skin – seen after the completion of the treatment of the kala-azar .

• Develops in 10% cases in India & 3% in Africa.

• Characterised by multiple , hypopigmented , erythematous macules involving the face and the trunk.

• Gradually progressed to non tender plaplaques and nodules .

• In Indian form PKDL appears after a latent period of 2 yrs,and may even persist upto 20 yrs.

PKDL

Epidemiology • Leishmaniasis found in 88 countries ( 16 developed countries and 72 developing coutries )

and in 5 continents of Asia , Africa, South America, North America and Europe .

• Annual incidence of the disease 600,000 cases per year .

• An estimated 12 million people are infected , and 350 million people are at risk, Of these approx. 25% are of VL

• India: Kala-azar primarily seen in endemic form in Bihar and West Bengal and parts of Uttar Pradesh. Sporadic cases have been reported from Tamilnadu, Pondicherry,Assam, Orissa, Gujrat .

• Annual kala-azar incidence peaked at 85 cases per 10,000 person-years in 2004 and fell to 46 cases per 10,000 person-years in 2007.

• There has been a resurgence of kala-azar in India , due to failure in successful implementation of the control programme and emergence of resistance of the parasite to commonly used antimonial drugs.

• In India , Kala-azar restricted to eastern part , whereas Cutaneous leishmaniasis restricted to western states and Rajasthan .

Leishmania tropica complex

• It includes L.tropica, L.major, and L.aethipica –all these causes Old world cutaneous leishmaniasis

• L.tropica causes oriental sore (classical form), also known as Aleppo button, ericho boil, bouton de Biskra, Delhi boil etc.

• Typically these lesions always ulcerates and heal spontaneously .

• Habitat :L.tropica and other species are found in humans ( Amastigote form in monocytes and polymorpho nuclear leucocytes ) and promastigote in sand fly and culture.

• They are not found in the peripheral blood or in the internal organs • Morphology : similar to L.donovani • Life cycle : similar to L.donovani except that the amastigotes resides in the large

mononuclear cells of skin .• Pathogenesis:bite of sand fly causes deposition of promastigotes on surface of the skin ,

at the site of inoculation promastigotes are phagocytosed by macrophages and transformed into amastigotes .

• Papule- in the early stage of the disease , and Ulcer –single or multiple , key pathological lesion in the skin ( oriental sore) .

Oriental sore

Types of Cutaneous leishmaniasis

• Two polar forms are found:

Diffuse cutaneous leishmaniasis – occurs in an anergic host with poor immune response ,papule without ulceration , satellite lesion around the papule ,mainly in face & extremities , chronic form lasts for 20 yrs or more .

• Leishmania skin test is negative .

Leishmaniasis recidivans : occur years after localised lesion healed , due to reactivation of dormant parasites or following new infection from a different species .

• usually in face , also causing destruction of nose .

• Skin test strongly positive , suggestive of exaggerated delayed hypersensitivity reaction .

• The condition is chronic and even persists for 20 years.

New world cutaneous leishmaniasis

• Caused by : L.braziliensis comple x( L. braziliensis, L. panamensis, L. guyanensis, L.peruviana)

• L. mexicana complex( L. mexicana,L. venezuelensis,L. amazonensis)

• Habitat : These are intracellular parasites of man .

• Found inside the macrophages of the skin and the in the mucous memb. of the nose and buccal cavity .

• Absent in either in the inernal organ or in the peripheral blood

• Morphology: similar to that of L.donovani • • Life cycle : similar to L.donovani , except that amastigotes are found in

RE cells and lymphatic tissue of the skin but not in the internal organs .

Pathogenesis • Pathogenesis of Oriental sore and Espundia are same except :• Lesion in espundia has a tendency to expand rapidly , with distinct margins

• Mucosal metastasis occurs in the mucosal leishmaniasis by L.braziliensis, it invades mucous memb . of mouth , nose , pharynx, and larynx either by direct extension or by blood

• Host immunity : development of marked cell mediated hypersensitivity reaction • The suppression of cell mediated immunity causes amastigotes to multiply and

spread in the skin causing diffuse cutaneous leishmaniasis

Different cutaneous manifestations

• L.braziliensis complex : • Espundia : most severe and destructive form of cutaneous

leishmaniasis, single or multiple ulcers are found in lower extremity • Pian bois: by L.guyanensis : ( forest jaws) single or multiple , painless,

dry persistent ulcers all over the body • Uta : by L.peruviana: single or multiple ulcers in the face, nasopharynx

not affected , heal spontaneously within 3 months to year

• L.mexicana complex : • Chiclero ulcer : (bay sore ) by L.mexicana : ulcers in the hand or head,

heals spontaneously within 6 months • Indolent nodular lesion : by L.venezuelensis• Cutaneous single sore type : by L.amazoniansis , sometimes causing

DCL

New world VL (AVL)

• Caused by L.chagasi in tropical South America (AVL)

• Mammalian hosts include human and domestic dog

• Lutzomiya vetulus is the vector

• Affected primarily the children ( similarity with L.infantum), potentially fatal if not treated

• Life cycle & morphology: same as L.donovani , amastigote can be found throgh out the body ( bone marrow, spleen, liver)and are not confined to the skin macrophages .

• Clinical disease: AVL is associated with malnourished children>5 yrs and young adults .

Laboratory Diagnosis

• Parasitic diagnosis: demonstration of Leishmania in different specimens confirms the diagnosis .

• Specimens:

• Spleen, bone marrow, liver, lymph node, and peripheral blood smear ,

• margin of the skin ulcer either by puncture of the raised nodule or aspiration of the

outer edge of the ulcer , Full thickness skin biopsy from margin of the ulcer,

• Slit skin smear

• Methods of examination :

• Microscopy

• Culture

• Animal Inoculation

• Immunodiagnosis

Lab. diagnosis• Microscopy;

• The amastigotes of L.donovani ( LD bodies) can be demonstrated in the smears of spleen, bone marrow, liver, lymph node, peripheral blood smear stained with Leishman, Giemsa, or Wright stain

• Brown –Hopps staining is a recent method shown to have a higher sensitivity compared to other staining techniques

• Splenic aspiration: is the most sensitive ( but there is a risk of haemorrhage)• Bone marrow aspiration : Sternum/ Iliac crest , safer • Liver biopsy:- not so safe

• LN biopsy: Positive in 60 % cases

• Peripheral blood smear : LD bodies can be seen in monocytes and neutrophils, Buffy coat

Brown-Hopps Method

• Fixation • 10% Buffered Neutral Formalin • Section • Paraffin, @ 6 microns • Staining Procedure • Deparaffinize and hydrate to distilled water. • Place slides on staining rack and pour on Crystal Violet Solution, 1%, Aqueous for two minutes. Rinse

in distilled water. • Mordant in Gram's Iodine for five minutes. Rinse in distilled water. • Differentiate in Cellosolve, until blue color no longer streams away from the section (approximately

5-10 seconds).

• Quickly rinse in distilled water. • Stain in Basic Fuchsin Solution, 0.5%, Aqueous for five minutes. Rinse in distilled water. • Gallego's Solution , for five minutes. (Differentiates and "fixes" the Basic Fuchsin). • Rinse thoroughly in distilled water and blot lightly to remove excess water (not to dryness). • Stain for three seconds in Tartrazine Solution . Immediately blot away excess, but not to dryness). • Cellosolve three changes, for six quick dips in each. • Xylene, three changes, for 10 dips each. Slides may remain in Xylene until ready for mounting. • Mount with Permount

Amastigote stage of Leishmania donovani. Showing Leishman-Donovan (LD) bodies in spleen smear of experimentally infected hamster. Many intracellular and extracellular

LD bodies are seen. x400

Culture:

• Tissue samples and aspirates are inoculated in:

• NNN( Novy ,McNeal,Nicole) media ( 2 parts of salt agar & One part of defribrinated rabbit blood) ,

• Schneider drosophila media( Insect cell culture media) ( 20 % foetal calf serum) , better than NNN

• Grace’s, and Mituhasi –Maramorosch media for demonstration of promastigotes ( Insect cell culture media)

• Incubated at 22°C-26°C for 1-4 wks.

• At the end of each week a drop of culture fluid is examined for promastigotes

Animal inoculation

• Intraperitoneal inoculation of chinese and Golden hamster may reveal parasites

• In a positive case amastigote can be demonstrated

Immunodiagnosis

• The hallmark of visceral leishmaniasis is hyperimmunoglobulinaemia, while in case of cutaneous and mucocutaneous leishmaniasis, the humoral immune response is extremely poor.

• Exploiting this host-parasite interaction, for the diagnosis of visceral leishmaniasis a number of antibody detection methods have been developed from time to time.

• Some of these tests include:• Non- specific tests and specific tests

• Indirect haemagglutination (IHA),

• Counter current immunoelectrophoresis (CCIEP),

• Immunodiffusion (ID) etc.

• These tests are cumbersome and lack sensitivity and specificity and hence not commonly used

Non- specific serological tests

• These test detects the hypergammaglobulinemia:

• Aldehyde test of Napier• Antimony test of Chopra

• Aldehyde test :done with pt’s serum , positive result shows jellification of a milky white opacity within 2-20 mins.

• Disadvantage – it shows false positive with sera from Schistosoma japonicum, African trypanosomiasis, multiple myeloma, cirrhosis of liver.

• It is always negative in cutaneous leishmaniasis

• Antimony test : a positive test is indicated by development of a white flocculent precipitate on addition of urea stilbamine solution to the patient serum

•

Specific immunological tests : 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 .

• The sensitivity of these tests varies extremely from as low as 28.493 to 86.6 per cent. 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.

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 per cent sensitive and 72-

100 per cent specific.• The method uses whole 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 long incubation time of 18 h 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.

• Recently, Schoone et al. have 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.

Enzyme linked immunosorbent assay (ELISA):

• ELISAis 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.

• An excretory, secretary and metabolic antigens released by L. Donovan promastigotes (Ld-ESM) into a protein free medium was used for the serodiagnosis of VL by ELISA. This antigen has been reported to be 100 per cent specific and sensitive, the PPV was 99.99 per cent and NPV was 95.45 per cent.

• The protein encoded by L. donovani gene B homologue (Ld-rGBP) , performing ELISA by using this antigen is reported to be specific for L. donovani infections only.

• Another recombinant protein rORFF of L. infantum origin has been developed by Raj et al. for diagnosis of VL in India.

• The ORFF protein is encoded in the LD1 locus of chromosome 35 of L. infantum. An ELISA based test using this antigen was found to be highly sensitive and specific.

• In addition, rK39 ELISA, has a high predictive value for detecting VL in immunocompromised persons, like AIDS patients78. This antigen is now commercially available in the form of antigen-impregnated nitrocellulose paper strips adapted for use under field conditions

ELISA

• Very recently, cloning and characterization of a recombinant antigen from an Indian isolate of L. donovani strain KE16 is done .

• The antigen (rKE16) is found to be 100 per cent sensitive and specific.

• In fact it has better sensitivity than rK39 which showed 98 per cent sensitivity for the diagnosis of Indian kala-azar and PKDL.

• It also showed 100 per cent concordance with rK39 in sera from leishmaniasis patients from China, Pakistan, and Turkey.

• This antigen has now been commercialized and has got tremendous potential for the serological diagnosis of VL worldwide.

Rapid antibody detection method

• 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 Span Diagnostic Limited (India) which uses Ld-rKE-16 antigen.

• Both are commercially available and are based on membrane filtration technology.

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.

• Recently, a latex agglutination test (KATEX) for the detection of leishmanial antigens in the VL patients’ urine has been developed.

• The test had 100 per cent specificity and sensitivity between 68-100 per cent.

Leishmanin skin test (LST): Montenegro test

• 0.2 ml of leishmania antigen ( containing 10^8 promastigotes of L.donovani in 1ml of 0.5% phenol saline ) is injected intradermally. Test is read after 48 to 72 hrs .

• Positive – area of erythema & induration of ≥5mm in D ,which heals in 14-25 days .It indicates prior exposure to leishmanial parasites .

• In kala-azar , it becomes positive usually only 6-8 wks after cure from the disease.

• 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.

Molecular methods

• A variety of nucleic acid detection methods targetingDNA 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, but limited to tertiary care hospitals and research laboratories.

• The important gene targets are 18S-rRNA, small subunit rRNA (SSU rRNA), a repetitive genomic sequence of DNA, the miniexon (spliced ladder) gene repeat, the b-tubulin gene region, gp63 gene locus, internal transcribed spacer (ITS) regions; micro-satellite DNAs such as maxi- and minicircles of kinetoplast DNA

Molecular methods

• Recently Martin-Sanchez et al. using PCR-ELISA found 24% asymptomatic individuals carrying Leishmania kDNA in their blood.

• It was pointed out that these individual could be potential source of transmitting the infections in the community.

• These authors also found a good correlation between the antibody titres, skin test positivity and PCR positivity.

• Bone marrow, lymph node aspirates, skin biopsy, skin scrape/exudates and blood samples have been used for PCR in several studies.

• The specificity of PCR on bone marrow aspirates has been reported up to 100% and sensitivity 80-93.3% as compared to 50-60% sensitivity of smear and culture examination.

• Also a modified form of PCR such as nested PCR has proved its predictive values in diagnosis of PKDL.

Molecular methods• The chronic CL patients are greatest diagnostic challenge In such

cases, PCR has been proved to be the most important tool for diagnosis. The sensitivity of PCR in CL has been reported 100 per cent.

• A recent development in PCR technology (fluorogenic probes and automation) takes care of non-specific amplification, speed and inter-operator variability.

• The real-time PCR is used qualitatively and quantitatively, as the

fluorescence is directly proportional to the number of amplicons, or in other words, the parasite load in the given specimen.

• The automization of real-time PCR with comprehensive portable units has made these tools field friendly.

• The multiplex PCR can be used whenever, double or mixed infections are suspected as in AIDS patients.

Summary of the Diagnosis

• Direct visualisation of amastigotes in clinical specimens is the diagnostic gold standard in regions where tissue aspiration is feasible and microscopy and technical skill are available.

• Diagnostic sensitivity for splenic, bone marrow, and lymph node aspirate smears is 95%, 55–

97%, and 60%, respectively

• Elsewhere, including epidemic settings,serum antileishmanial immunoglobulin G in high titre is the diagnostic standard, primarily with direct agglutination tests or other laboratory-based serological assays.

• Freeze-dried antigen (refrigeration not needed) and rapid detection of anti-K39 antibody with fingerstick blood in an immunochromatographic strip test have advanced field serodiagnosis.

• In symptomatic patients, anti-K39 strip-test sensitivity is high (90–100%)and while specificity might vary by region this test can safely substitute for invasive diagnostic procedures in Indian visceral leishmaniasis and is useful in PKDL.

• Testing urine for leishmanial antigen or antibody is a new approach.

• In a central laboratory, clinical samples can be cultured for parasite isolation and leishmanial DNA is readily detectable by PCR testing, including in peripheral blood and serum.

Giemsa-stained splenicaspirate smear, showing clumped mononuclear cells and numerous amastigotes . OriginalMagnification X500.

Serodiagnosis of kala azar by anti-K39 antibody detection in immunochromatographic strip test. 5 min after application of one drop of fingerprick blood and buffer, right-hand strip shows second pink band (arrow) indicating presence of anti-K39. Left-hand strip shows a negative result (no second band).

TREATMENT

• The drugs of choice today are the same compounds that were used in the early 1900s and their mechanisms of function are not completely understood. They are all extremely toxic, and the patient must be monitored closely during treatment.

• Pentavalent antimony coumpounds, derived from the heavy metal antimony (Sb), are the drugs of choice for treating cutaneous and visceral Leishmaniasis. The recommended dosages follow:

•

• Studies have shown that longer courses prove more effective in preventing relapse, however they also increase the risk of drug-related toxicity. Further complicating the issue is that parasite resistivity seems to be increasing and relapse rates in some areas have been reported as high as 50%-70% with mucocutaneous leishmaniasis and 10% with visceral leishmaniasis.

• Side effects include: Cardiotoxicity, reversible renal insufficience, pancreatitis, anemia, leukopenia, rash, headache, abdominal pain, nausea, vomiting, arthralgia, myalgia, thrombocytopenia, and transaminase elevation.

Disease Dose Duration

Cutaneous leishmaniasis 20 mg Sb/kg/day 20 days

Mucocutaneous leishmaniasis 20 mg Sb/kg/day 28 days

Visceral leishmaniasis 20 mg Sb/kg/day 28 days

Treatment

• Amphotericin B has been shown to be more effective than pentavalent antimony in vitro, but has not been used extensively in the past due to extreme toxicity.

• However, more recent developments have made Amphotericin B less toxic and more useful. It has also become useful in treating strains of visceral leishmaniasis causing organisms that are resistant to pentavalent anitmony.

• Amphotericin B cannot be used in the treatment of mucocutaneous leishmaniasis because very large doses are required.

• Side effects include: fever nausea, vomiting malaise, anemia, phlebitis, hypokalemia, hypomagnesemia, and nephrotoxity.

• Pentamidine isothionate is a general anti-parasitic drug that has demonstrated some success with the treatment of viceral leishmaniasis. Unfortunately the cure rate has declined from the early 1980s to the 1990s due to resistance of Leishmania to pentamide. Nonetheless, it can be used as a second-line agent in the event that the pentavalent antimony compounds fail.

• Side effects include: nausea, vomiting, abdominal pain, and headache.

Treatment

• Other Agents:

• The following drugs and therapeutic approaches seem promising and are currently being studied, but have not been approved for general use.

• Cytokine therapy: Cytokines such as interferon gamma and granulocyte macrophage colony-stimulating factors are being used in combination and with pentavalent antimony in attempts to enhance reponse.

• Miltefosine: An oral drug for visceral leishmaniasis is showing progress in studies. Apparently it is less toxic and exhibits higher cure rates than traditional drugs.

• Furthermore, a cheap, orally administered treatment would be extremely beneficial for the people most affected by leishmaniasis

VACCINES:

• Currently there are no drugs or vaccines for the prevention of Leishmaniasis.

• Leishmaniasis is an intracellular infection.

• Ideally a vaccine would illicit a strong Th1 response as a Th2 response has been implicated in chronic, non-healing disease.

• Live-attenuated candidates have been proven to create this kind of response, but the dangers associated with them have hindered their use.

• With the help of adjuvants and cytokines, whole-killed vaccines seem show great promise.

Vaccines

• Whole-killed Leishmaniasis: Currently, the most successful vaccine attempts in humans has been achieved with whole-killed leishmania promastigotes.

• Phase III clinical trials in the Middle East and South America with an autoclaved Leishmania and BCG have proven to be effective at reducing the incidence of cutaneous leishmaniasis.

• Reported efficacy rates range from 18% to 78% (Sharifi, 1998; Antunes, 1986). Evidence also suggests that the Leishmania major creates a certain degree of cross-protection to Leishmania donovani, the parasite implicated in the deadly visceral leishmaniasis (O'Daly JA, 1993).

• The vaccine seems to boost IFN-gamma and T-cell production, inducing a strong Th1 response. This strong Th1 response may also explain cross-protection to another intracellular parasite Trypanosoma cruzi (Araujo, 1999) and it may account for its proven therapeutic qualities for individuals and mice infected with cutaneous Leismaniasis.

• It has been shown to have similar cure rates as standard antimonial regimens with fewer side effects (Convit, 1987).

• Unfortunately, the autoclaved parasite shows decreasing efficacy with time. Studies of efficacy with thiomersal-preserved and non-autoclaved preparations have shown to keep better (De-Luca, 1999).

Vaccines

• Live-attenuated: Historically, protection from cutaneous leishmaniasis was achieved by taking the pus from a healing lesion and innoculating a naive recipient in an inconspicuous location.

• This crude version of a live-attenuated vaccine conferred protection upon the host, however serious disease was occasionally a complication.

• Both protection and disease stem from the parasites ability to get inside the cells and stimulate a cell-mediated T-cell response.

• Radioattenuated promasitigotes and biochemically altered Leishmanial parasites have proven to confer very good protection in mice and hamsters without adjuvant (Rivier, 1999).

• Unfortunately, live cultures of Leishmaniasis are hard to sustain and there is always the concern of reversion to a virulent strain.

• Synthetic Peptide:

A single synthetic T-cell epitope from gp63 administered with Th1 stimulating poloxamer 407 conferred protection against Leishmania major in BALB/c mice (Spitzer, 1999).

• Other: Infection of mice with adenovirus expressing IL-12 skews the immune response in a Th1 direction preventing leishmaniasis in susceptible mice (Gabaglia, 1999).

• Antibodies produced against Liposphoglycan (LPG) may prove to be effective in limiting the transmission of cutaneous leishmaniasis in its vector Phlebotomus dubosqi (Tonui, 1999).

• To date, several different approaches to antileishmanial vaccine have been tested.

• First generation vaccines composed of whole killed parasites have been proposed as both prophylactic and therapeutic vaccines.

• In theory, these vaccines should be easy to produce at a low cost in endemic countries; however, standardization of cultured parasite-derived vaccines is a drawback in the way to their registration.

• In general, the whole-cell, killed vaccines have been rather poorly defined and variable in potency, hence they have rendered inconclusive results.

• Nevertheless, the trials completed so far demonstrated their good safety profile, and despite poor prophylactic outcomes, showed encouraging results as therapeutic vaccines in South America and Sudan.

• Most of the vaccine studies concentrate on the second generation vaccines consisting of recombinant proteins, poly-proteins, DNA vaccines or dendritic cells loaded with peptides derived from leishmanial antigens.

• A variety of different molecules has been tested to date, and these included antigens such as surface expressed glycoprotein leishmaniolysin (gp63) delivered by a plethora of immunization regimens, however, promising findings from animal models were overshadowed by mostly negative T cell responses in humans.

• Another vaccine candidate has been a GPI-anchored membrane protein gp46 or Parasite Surface Antigen 2 (PSA-2), that belongs to a gene family present in all Leishmania species except L. braziliensis.

• Immunization with the native polypeptides derived from promastigotes protected mice against infection,but vaccination with a recombinant protein derived from either promastigotes or amastigotes protein showed lack of protective efficacy.

• Similarly, DNA vaccination conferred protection in mice when used as either prophylactic or therapeutic vaccines.

• Another extensively tested antigen is the Leishmania homologue for receptors of activated C kinase (LACK) that is expressed throughout leishmanial life cycle.

• Immunization with LACK appears to promote the expansion of IL-4 secreting T cells skewing the response towards detrimental Th2 responses.

• However, susceptible BALB/c mice immunized with LACK had the ability to control a subsequent infection with L. major.

• To date, the protective efficacy of LACK has been mainly demonstrated in the L. major model, and LACK failed to protect against visceral leishmaniasis.

• Several other antigens from different species have been tested in animal models.

• These include amastigote cysteine proteases (CP),cysteine proteinase A2 and

amastigote membrane proteins P4 and P8, kinetoplastid membrane protein-11 (KMP-

11), amastigote LCR1, hydrophilic acylated surface protein B1 (HASPB1), leishmanial

antigen ORFF, acidic ribosomal protein P0, paraflagellar rod protein 2 (PRP-2), NH36, a

main component of the fucose-mannose ligand and proteophosphoglycan (PPG).

• In addition, molecules such as ATP synthase alpha chain, beta-tubulin and heat shock 70-related protein 1 precursor have been recently identified as novel vaccine candidates.

Vaccines

• To date, only one second generation vaccine, Leish-111f, has been assessed in clinical trials.

• Leish-111f is a single polyprotein composed of three molecules fused; the L. major homologue of eukaryotic thiol-specific antioxidant (TSA), the L. major stress-inducible protein-1 (LmSTI1) and the L. braziliensis elongation and initiation factor (LeIF).

• Initial immunisation trials in mice demonstrated that Leish-111f was able to protect mice against L. major and L. amazonensis infection.

• There is some evidence that the Leish-111f vaccine can also induce partial protection against visceral leishmaniasis in animal models, however, Leish-111f failed to protect dogs against infection and did not prevent disease development in a recent Phase III trial in dogs.

• A slightly improved version of the original construct, Leish-110f, has also been tested in dogs as a therapeutic vaccine in combination with chemotherapy and led to reduced number of deaths and higher survival probability.

• Human Phase I and II clinical trials (safety and immunogenicity) of Leish-111f have been completed over the past few years in Brazil, Peru and Columbia, and Phase I trial has been conducted in India (http://clinicaltrials.gov).

Reference centre • RAJENDRA MEMORIAL RESEARCH INSTITUTE OF MEDICAL SCIENCES

PATNA - 800007 (Established in 1981)• Phone: 0612 -2631565 Fax: 0612 -2634379 Email : rmrims@rmrims.org.in,

dirrmris@sancharnet.in Website : http://www.rmrims.org.in • Scope of Activities: • Situated at Patna in Bihar State, the Rajendra Memorial Research Institute of Medical

Sciences (RMRIMS) conducts research on various aspects of Visceral Leishmaniasis, also known as Kala-azar, and HIV/AIDS. Tuberculosis is now being included. RMRIMS is one of the designated ICMR centres for bio-medical informatics.

• Thrust Areas:• PCR based diagnosis • Studies on VL-HIV co-infection • Clinical trials • Cost effective integrated vector management • Leishmania repository• Role of cytokines in responsive and unresponsive patients• Immuno-pathology of PKDL patients• Innate immunity in malnutrition • Database design of leishmania parasite • Routine Biochemical and Hematological Diagnosis and Treatment of VL/PKDL/HIV• HLA Typing of VL Patients • Studies on Drug Resistance Mechanism in Genomic and Proteomic Level

THANK YOU

Refferences

• Toplay & Wilson’s Parasitology 10th edn.• Diagnostic medical parasitology –Garcia 4th edn.• Parasitology by K.D.Chatterjee-12th edn.• Text book of medical parasitology –S.C .Parija-3rd edn.• Medical parasitology –D.R.Arora,B.Arora- 2nd edn.• Internet sources

Vaccines

• Leishmania parasites are transmitted from one host to another during the sand fly bite as a suspension in sand fly saliva.

• Sand fly saliva contains an array of molecules able to interfere with the host immune responses,

• therefore, immunity against saliva components may indirectly enhance anti-leishmanial immunity.

• Prior exposure of mice to bites of uninfected sand flies conferred protection from L. major infection.

• Immunization with molecules present in saliva, such as maxadilan or a 15 kDa protein, SP15 also induced protection against cutaneous leishmaniasis.

• More recently, it has been shown that vector salivary proteins, in particular LJM19, protect hamster from VL,and immunization of dogs with salivary antigens led to the development of high IgG2 antibody levels and significant IFN-γ production.