LEISHMANIASIS

DR. DEBARATI BANERJEE

 HISTORY
Pre Incan pottery depicted skin lesions resembling CL
Inca period texts (15th - 16th centuries) Valley Sickness
or Andean Sickness or White Leprosy

 1756, Alexander Russell: clinical description-Aleppo Boil

 1898: Peter Borovsky → protozoal nature

 1900:Dr William Leishman → parasite from spleen of patient who

died of Dumdum fever

 1903: CharlesDonovan → “Leishman Donovan” bodies

  1903:Ronald Ross → named Leishmania

donovani.

 1922: Dr U.N Brahmachary →

Discovered Urea-Stibamine
-
 The Parasite
Phyllum Sarcomastigophora

Order Kinetoplastida

Family Trypanosomatidae

Genus Leishmania
Subgenus Disease Old World Species New World Species

LEISHMANIA VL L.donovani L.chagasi

L.infantum

L.archibaldi

CL L.major

L.tropica , L.killicki

L.ethiopica L.mexicana complex

(L.mexicana,L.amazonensis,
L.venezualensis,L.garnhami,
L.pifanoi)

VIANNIA L.braziliensis ,L.peruviana

L.guyanensis ,L.panamensis

L.liansoni

Lnaiffi

MCL L.braziliensis

L.panamensis
 2 morphological forms

Amastigote stage
 Aflagellate state
 Intracellular in RE cells of the mammals
 Round to oval,2-4μM
Ultra structurally
Nucleus: central
Flagellar pocket: endocytosis, exocytosis,
anchoring the flagellum
Axoneme(Flagellum): nonfunctional
Kinetoplast : mitochondrial DNA
Vacuole :clear unstained space alongside axoneme
Microtubules: serves as cytoskeleton
Glycosome: contains the glycolytic enzymes
Promastigote stage
 Flagellate , Infective stage
 Found in Sandfly and cultures
 Elongated spindle shaped ,8-15
μM
Ultra structurally
 Kinetoplast transverse & more
anterior than nucleus
 Flagellum –arises from anterior
end
 Paraxial rod inside flagellum
plays role in flagellar motility
 Desmosomal plaques anchor
flagellum to cell body
 VISCERAL LEISHMANIASIS
Distribution of VL Worldwide
 Found in 47 countries
 90% of all VL: Bangladesh, Brazil, India, Nepal and Sudan
 Annual incidence: 500,000 cases of VL
 OLD WORLD VL
 Caused by L.donovani, L.infantum ,L.archibaldi & rarely L.tropica

 L.donovani: Classic VL- S.Asia: India ,Nepal, Bangladesh

East Africa(Sudan, Somalia, Ethiopia, Kenya, Uganda)
Sporadic-China,Pakistan

 L.infantum: infantile VL- Mediterranean basin, Middle East,

Central & S.W.Asia, China, N & Sub-Saharan Africa
 L.archibaldi: in Africa

 L.tropica : Atypical VL- Middle East, Saudi Arabia, India, N.Africa

 VL important opportunistic disease among AIDS pts

 In Mediterranean Europe (Spain, France, and Italy)- 70% of adult VL cases

associated with HIV co-infection
New world VL
 Also called American VL (AVL)
Most of Central and South America
Major periurban outbreaks of VL reported from N.E Brazil

AGENT:
L.chagasi →Visceral and Cutaneous manifestations

Contined debate about L .chagasi being not a separate

species & same as L.infantum ,probably introduced into New
World by early exploration
SITUATION IN INDIA
India worst affected country

40-50% of global burden

INDIA: 15538 cases and 47 deaths

by VL (2010)

Endemic states in Eastern India: Bihar,

Jharkhand, West Bengal, Uttar Pradesh

Estimated 165.4 million population at risk

in 4 states

Sporadic cases reported from Tamilnadu,

Pondicherry, Gujarat,
Punjab, Assam & Orissa


 VECTOR FACTORS
 Female sandflies of genus Phlebotomas(Old World)& Lutzomyia
(New World)
 Small hairy insects, Family-Psychodidae
subfamily: Phlebotominae
 Females → blood meal before laying eggs
 BLOCKED sandflies highly infectious
 Pool feeders

 Vectors of Leishmania donovani complex are:

 Indian Vector - P.argentipes

 Mediterranean - P.perniciosus, P. ariasi
 Sudan - P.orientalis
 China - P.chinensis,P. alexandri
 Latin America - L. longipalpis
 Kenya – P. martini
 RESERVOIR OF INFECTION
In areas of L.infantum prevalence i.e
Mediterranean areas –
China – Dogs,Foxes,Jackals
Middle East –

In areas of L.donovani prevalence i.e

India- ●Man is the only reservoir
Middle East- ●PKDL cases
Africa- (maintain the parasite between
Parts of China,Europe,S.A- epidemics)

In areas of L.chagasi prevalence i.e

Latin America- Dogs & Foxes
 HOST FACTORS
a. AGE: all age groups susceptible, peak age 5-9 yrs
b. SEX: males > females
c. POPULATION MOVEMENT
d. SOCIO ECONOMIC STATUS: poor
e. OCCUPATION: farmers, foresters, miners
f. IMMUNITY: -Immunosuppression - predispose to VL
-Recovery - lasting immunity

ENVIRONMENTAL FACTORS

A. ALTITUDE: confined to plains

B. SEASON: during & after rains
C. RURAL>URBAN AREAS
 MODE OF TRANSMISSION
 Natural Transmission- bite of the infected female phlebotomine sand fly

 Other methods of Transmission:

 Congenital infection
 Blood Transfusion
 Accidental inoculation of Cultures
 Sexual Transmission
 Sharing of contaminated needles & syringes
(high prevalence of L.infantum/HIV co-infection in iv drug addicts
in Southern Europe)
LIFE CYCLE
Development in Sandfly Host
 TRANSMISSION FROM SANDFLY TO MAMMALIAN HOST
 10-1000 metacyclic promastigotes intradermally per infective bite

 Infectivity is enhanced by

 sandfly saliva: contains complex mixture of pharmacologically active

compounds e.g Maxadilan,a potent vasodilator ,
anticoagulant, platelet inhibitor & immunosuppresant

 PSG(promastigote secretory gel) :

 produced by Leptomonad forms
 contains mucin like proteophosphoglycan
 parasite/PSG blocks anterior midgut → difficulty in feeding →
Blocked sandfly →mutiple probing →enhances transmission
 PATHOGENESIS
 Interaction of promastigotes with skin macrophages
↓
 Promastigotes activate complement via Alternative pathway
↓
 Activated ‘C’ components C3b & iC3b→ Bind to receptors - gp63 & LPG on
promastigotes
↓
 These receptors bind with “C” receptors CR3 & CR1 present on macrophages
 ↓
 Phagocytosis of promastigote in a phagosome→fusion with lysosomes
→transformed into amastigote →multiply by binary fission
↓
 Macrophage ruptures →releases large no amastigotes into circulation
↓
 Free amastigotes invade RES of spleen, liver, bone marrow.& L.node
PARASITIC FACTORS IN PATHOGENESIS

1) Invasive/evasive determinants

 (i) Leishmania–macrophage attachment

 (ii) entry of Leishmania into macrophages
 (iii) intramacrophage survival
 (iv) differentiation , intracellular multiplication of amastigotes

2) Pathoantigenic determinants
 conserved structural or soluble cytoplasmic proteins
 contain immunogenic B-cell epitopes

HOST FACTORS
 host immune responses
 host genetic factors
 host nutritional status
Invasive/evasive determinants
 PSP(Promastigote surface protease)/gp63
 size 63000kDa,Zinc metalloprotease ,an Ectoenzyme
 Proteolytic activity against lysosomal enzymes help transformation
 Binds promastigotes to macrophages via complement receptors
 Facilitation of parasite migration through host tissues

 GLYCOPHOSPHATIDYLINOSITOL(GPI)
 Found on the surface on both promastigotes & amastigotes
 Protect amastigotes against hostile environment of phagolysosome

 CYSTEINE PROTEASES(CPs)
 facilitates survival and growth of parasites in mammals by
 destruction of host proteins,

 evasion of the host immune response

 LIPOPHOSPHOGLYCAN(LPG)
 Contains 4 covalently linked structural components:
- phospholipid tail -Glycan Core
-Backbone of Phosphodisaccharide Repeats - Cap
 Interferes with complement-mediated lysis by steric hindrance
 LPG fixes complement→ provides ligands for binding with macrophage C
receptors → stimulates phagocytic uptake of promastigotes
 Inhibitory effect on macrophage protein kinase C
 Efficient scavenger of toxic o2 radicals
 Protects against hydrolytic enzymes in phagolysosome

HOST IMMUNE RESPONSE

 Evidence of both protective & disease enhancing immune responses
 Cytokines & chemokines play key roles
 INNATE IMMUNITY
 Leishmania parasites are first confronted with the host’s innate immune response
↓
 Control of infection mediated by intrinsic capacity of macrophages to become
infected by promastigotes & amastigotes
↓
 produce (IL-12) and other proinflammatory cytokines (TNF - ∞,IL-1)
↓
 IL-12 has a key role in activation of NK cells to secrete IFN-γ
↓
 IFN- γ and TNF- ∞ activates infected macrophages
↓
Intramacrophagic radical oxygen (ROS) is produced
 increased production of by the NADPH oxidase complex
NO produced by the inducible (iNOS)
↓
 are potent anti-Leishmania molecules
↓
 causes parasite death
 ACQUIRED IMMUNITY
 infected macrophages and dendritic cells present parasite peptide Ag to T cells by MHC
↓
presence of IL-12

Activation of CD8+ Activation of CD4+ Activation of CD4+ T helper

Cytotoxic T cells T helper Th1 subset Th2 subset
↓ ↓
Parasite death Th1 cells secrete cytokines Devolopment of Produce cytokines
IL-2, IFN- γ ,GM-CSF humoral response IL4,IL5,IL10,IL13.
↓ ↓ ↓
induce cell-mediated Production of Ab inhibit both
immune responses -Th1 immune response
↓ -macrophage
activation
induction of macrophage
microbicide activities and
activation of cytotoxic T cells
IMMUNOLOGICAL DETERMINANTS IN PARASITE MULTIPLICATION
 CLINICAL FEATURES OF V.L
 Disease characterized by
 Fever , Splenomegaly ,Hepatomegaly, asthenia, muscle wasting
 Anaemia: -normochromic,normocytic
 Leucopenia,Thrombocytopenia ,↑ γglobulin

 Clinical Forms :
 Asymptomatic and subclinical infections
 Infantile visceral leishmaniasis due to L. Infantum
 L.Donovani Visceral leishmaniasis
 Viscerotropic Leishmaniasis

 L.tropica infection
 Chronic low grade fever, malaise, fatigue, mild splenomegaly
 1st described in U.S militants in Gulf War in 1990-91, later in Brazil.Italy
Post kala-azar Dermal Leishmaniasis

Feature East Africa Indian Subcontinent

Most affected country Sudan Bangladesh

Incidence among patients with 50% 2%
VL
Interval between VL and PKDL During VL to 6 months 6 months to 3 years
Age distribution Mainly children Any age
History of prior VL Yes Not necessarily
Rashes of PKDL in presence Yes No
of active VL
Treatment with sodium 2–3 months 2–4 months
stibogluconate
Natural course Spontaneous cure in majority Spontaneous cure not
patients reported

Dermal lesions of PKDL are

• Hyperpigmented /hypopigmented macules
 Erythematous Patches,
 Yellowish Pink Nodules
 Found on face,trunk, extremities,oral m.m,& occasionally genitals
 Visceral Leishmaniasis & HIV Co-infection
 Reported from 35 countries
 HIV increases risk of developing VL severalfold in endemic areas
 VL -opportunistic infection in late stage HIV ,CD4<200/ml

 Atypical presentation,CD4<50/ml
 Involvement -extensive G.I tract → present with chronic diarrhea
- lung & pleura → Pleural Effusion
- Bone Marrow → Aplastic anemia

 Co infected patients →parasites in skin & blood buffy coat(88%)

 Diagnosis → parasitological
→Serodiagnostic tests are commonly negative
 Rx-Liposomal AmB is the DOC -both for primary treatment & relapses
-AntiRetroviral therapy cornerstone for the management of HIV/VL
co-infection
LABORATORY DIAGNOSIS

DIAGNOSIS
 PARASITOLOGICAL DIAGNOSIS

Specimen
Splenic aspirate (most sensitive)
Bone Marrow aspirate (most commonly used)
Liver biopsy
Lymph node biopsy
Peripheral Blood & buffy coat

1)MICROSCOPY - Demonstration of Amastigotes

 Stains used: -panoptic MayGrunwald Geimsa stain
-Leishman stain, Wright stain
-Brown Hopps stain-Recent method-↑ sensitivity
-Immunohistochemistry

 Amastigotes detected using mouse anti L. amazonensis immune serum,a

Streptavidin peroxidase conjugate and Amino-ethyl carbazole (AEC) as
chromogen
Criteria for grading of parasitemea (WHO)
Grade Average parasite density
6+ >100 parasites/fields
5+ 10-100 parasites fields
4+ 1-10 parasites fields
3+ 1-10 parasites/ 10 fields
2+ 1-10 parasites/ 100 fields
1+ 1-10 parasites 1000 fields
0 0 parasites 1000 fields

Splenic B.M Liver L.N Blood

aspirate aspirate aspirate aspirate

SENSITIVITY 80 – 98% 60-85% 50 -75% 40 – 50% 0-30%

SPECIFICITY 100% 100% 98% 95% 100%
DEMONSTRATION OF MACROPHAGE FILLED
LEISHMANIA AMASTIGOTES /L.D BODIES

Geimsa stained Splenic Smear Geimsa stained Bone Marrow smear

 ISOLATION BY CULTURE
Culture media for axenic culture
 BIPHASIC MEDIUM
 NNN medium(Novy-MacNeal-Nicolle)
 Evan’s modified Tobie’s medium
 SOLID MEDIA(J.C.Ray)
 LIQUID MEDIA
 HO-MEM media
 Schneider’s Drosophila medium
 Grace’s insect tissue culture medium
 Mituhasi-Maramorosch media

 Strain identification by: Isoenzyme electrophoresis

Monoclonal antibodies& DNA probes
 Good Sensitivity and Low Sensitivity in case of
specificity. subclinical infection.
 Allows parasite Cumbersome
identification Time consuming
Invitro sensitivity can be Not suitable for field
carried out conditions
Requires highly trained
personnel.

Splenic B.M Liver L.N Blood

aspirate aspirate aspirate aspirate
SENSITIVITY 70 - 98% 40-50% 50 -75% 40 – 50% 0-30%

SPECIFICITY 100% 100% 98% 95% 100%

 M0LECULAR METHODS
DNA Hybridisation Assays
-large no of samples , quickly , efficiency
-samples like blood spots , splenic & B.M aspirates can be
processed

Recently Molecular Probes with high sensitivity & specificity

devoloped –
-Kinetoplast DNA (kDNA)
-Ribosomal RNA (rRNA)
-Mini Exon Derived RNA(med RNA)
-Genomic DNA repeats
Recently, a chemiluminscent hapten digoxigenin labelled
kDNA probes developed
 PCR - A RECENT DIAGNOSTIC TOOL
 highly sensitive & specific technique
 detect parasite in contaminated samples or cultures
 Relative promptness of result
 species identification
 differentiate between present & past infections

 Several TARGET sequences have been used for PCR

 e.g of targets ►Genomic DNA sequences:

►rRNA gene sequences :
18s rRNA
SSU-rRNA
med rRNA

►kDNA(conserved & variable regions)

 PCR …….CONTD
 Samples :VL – Bone marrow aspirate
splenic aspirate
Lymph node aspirate
Blood
PKDL – slit skin smears
 Recently various PCR based assays are in vogue:

1)PCR-SHELA (PCR- solution hybridization enzyme linked

assay)
-diagnose L.donovani infections in India , Kenya & Brazil
- 90% sensitivity & 100% specificity( Nuzum et al)
- epidemiological survey tool in central America presence of L.chagasi
-microtitre plates ,biotin labeled probe & spectrophotometer used
-suitable for field use,large no samples
-
2)PCR-SSCP : - detection of sequence variation of rRNA genes
within the L.donovani species
- Does not need prior cultivation of the parasite

3) Fluorogenic real-time PCR:

-used for post therapeutic follow up & for detection of
relapses among HIV +ve pts using SSU-rRNA gene target
-quantification of parasitic burden

4)ULTRASENSITIVE PCR:
-In VL allows detection of asymptomatic carriage in man
-Hence defines parasitaemia threshold above which
symptoms are likely to appear
 IMMUNO-DIAGNOSIS
NON SPECIFIC SEROLOGICAL TESTS
 Detection of Hypergammaglobulinaemia
1) Aldehyde (formol gel) Test (Napier) :
 +ve test - jellification of contents in 2-20 mins
 test –ve till 3 months after infection
 remains positive till 6 months after cure
 False +ve results in African trypanosomiasis, malaria and schistosomiasis
malaria.tuberculosis,multiple myeloma & cirrhosis of liver

2) Chopra’s Antimony Test: Not used nowadays

 Complement Fixation Test

- Antigen → W.K.K (tubercle bacilli), Ag from Kedrowsky AFB
- Becomes +ve three weeks after infection
- False +ve in L. leprosy, Pulm.T.B, Tropical pulm eosinophilia
 SPECIFIC SEROLOGICAL TESTS
ANTIBODY DETECTION

Indirect Fluorescent Antibody Test

 Detection of anti-leishmanial antibody using fixed promastigotes

 Test based on demonstration of Ab in the very early stages of infection

and become undetectable six to nine months after cure

 Titers >1:20 are significant and >1:128 are diagnostic

 Cross reaction with trypanosomal sera (overcome by

using Leishmania amastigotes as the antigen)
 DIRECT AGGLUTINATION TEST
 Use of whole, stained promastigotes either Relative long incubation time of
as a suspension or in a freeze-dried form 18 hours
which is heat stable
 Inexpensive & easy to perform Need for serial dilutions of serum
 Ideal for field & laboratory use No prognostic value
 Whole blood, serum or plasma,spotted
Remain positive for several years
blood can be used as samples after cure
 Serum titres of 1:3200 are considered +ve
Fast Agglutination Screening Test (FAST)
 Need of only 1 serum dilution
 Rapid: results available in less than 3 hours
 ELISA
Sensitivity and specificity greatly influenced by the antigen
used.
Earlier antigens :
 SOLUBLE LEISHMANIA Ag (SLA): Prepared from
promastigotes of L.infantum

R ecombinant antigens : rK39

rK39 ELISA
100 % sensitive and 97 % specific in diagnosis of VL and PKDL
ADV: -predictive of onset of disease manifestation in VL
patients
-directly correlate with active disease
-have potential in monitoring the chemotherapy
- predicting the clinical relapse
rK39 RAPID DIPSTICK TEST
Principle :-membrane pre-coated -rK39 on the test line region
- chicken anti-protein A on control line
-serum sample reacts with the Protein A-colloidal gold
conjugate
↓
migrates immunochromatographically
-captured by a line of rK39 antigen in test area→ produces a
pink line

- migrate to the immobilized chicken anti-protein A region,

- a red line on the control line region →sufficient sample
volume , proper flow & control for the reagents.
Interpretation of Results

+ve -ve
Result Result

An Invalid Result
•No lines appear at either the
control or test line areas.
• control line absent, but a test
line is seen.
 IMMUNOBLOT ANALYSIS
 detect antibodies against specific Ag according to Leishmania species
 more sensitive technique than IFAT or ELISA
Advantages:
 Confirmation of diagnosis
 Useful for follow-up of patient during treatment
Disadv: time consuming, technically cumbersome, expensive

Other tests
 Indirect haemagglutination (IHA)
Counter-current immunoelectrophoresis (CCIEP),

Leishmanin skin test

-measures delayed-type hypersensitivity
- acute VL –ve,turns +ve several months after clinical cure
 ANTIGEN DETECTION
 Recently latex agglutination test (KATEX) devoloped to detect
leishmanial antigen in urine of pts with VL
 test uses latex particles sensitised with Ab raised against L.donovani Ag
 urine sample boiled for 5 mins to avoid false +ve rxns with the latex reagent
 50μl sample mixed with 1 drop of the latex reagent
↓ 2 mins
If agglutination → test +ve for VL

ADVANTAGES
 Sensitivity :68 - 100% , specificity : 100 %
 Indicates treatment failure
 Detects VL in HIV coinfected patients
 TREATMENT OF VL
1) Pentavalent Antimonials (SbV) :
 Currently used:Sodium Stibogluconate,Meglumine Antimoniate

 DOC of VL in East Africa , Latin America & Mediterranian

 no longer recommended in India, where >50% of cases are now resistant.
2)Amphotericin –B Deoxycholate
 Polyene antibiotic used in the treatment of
 severe VL, MCL ● Sb resistant VL in India
 Treatment failure ● Relapses
3) Lipid-associated amphotericin B
Liposomal amphotericin B : DOC for the treatment of VL in North America
Adv: ♦ drug preferentially taken up byR.E tissues →↑es therapeutic index
♦ less toxic
♦ large amount of drug can be delivered over a short period
4) Miltefosine
an alkylphosphocholine, oral antineoplastic agent
first oral compound approved for Rx of VL
5) Paromomycin (Aminosidine ):
 latest drug registered for VL in India since 2002
efficiently used for VL as monotherapy and in combination with
Sb
6) Interferon γ
was successfully used in combination with antimonials in the
treatment of VL
use of IFNγ remains limited to date in spite of efficient clinical
trials
 PREVENTION & CONTROL
 Individual Prevention
 insect repellents containing DEET and permethrin applied to clothing
 insecticide-impregnated fine-mesh bed nets
 No CHEMOPROPHYLAXIS or IMMUNOPROPHYLAXIS for travelers

 Community –based prevention

 Vector Control: -Destruction of breeding sites
- Insecticide spraying

 Reservoir control:
 Anthroponotic transmission : case identification and treatment

 Zoonotic Transmission : - insecticide-impregnated collars for dogs

(deltamethrin-impregnated collar)
-treatment of infected domestic dogs,
-culling of street dogs
THANK
YOU

HAVE
A
NICE
DAY