zoology

1. Current Strategies in
Diagnosis & Treatment
of Leishmaniasis
Dr. Sayan Chakraborty
JR-3, Dept. of Tropical Medicine,
School of Tropical Medicine, Kolkata
Email: dr.sayan@gmail.com

2. Epidemiology
• Neglected tropical disease
• Incidence: 2 million/year
• Population exposed: 350 million
• Types: VL; PKDL; CL; MCL
• Endemic areas:
 VL: Indian subcontinent, East Africa, South America
(Brazil), Mediterranean region, Middle east, Central Asia,
China
 PKDL: Indian subcontinent
 CL: Middle east, Pakistan, Brazil, Columbia, Algeria
MCL: New world only (South America)

3. Leishmania Parasite

5. Transmission
• Routes:
 Bite of infected Sandfly
 Injection drug users
 Congenitally
 Blood transfusion
 Organ transplant
 Lab accidents
 Contact with active lesions of CL
• Vectors:
 Phlebotomus
 Lutzomyia

6. Transmission
Reservoirs
L. donovani Humans
L. infantum Dogs, wild foxes, crab-eating foxes, jackals,
wolves, racoon dogs
L. major Great gerbill, Fat Sand Jird (Africa)
L. ethiopica Hyraxes
L. guyanensis
L. panamensis
Sloths
L. amazonensis Spiny rat
L. mexicana Climbing rat
L. laisoni Paca
L. naiffi Armadillo

7. Leishmania Life Cycle

8. Pathogenesis
 VL: Disease of mononuclear phagocytic system
• Affects spleen, L.n. & bone marrow (rarely intestine, lung,
skin)
• Granulomata by proliferation of activated macrophages
• ↑ IL-1, IL-6, IL-8, IL-12, IL-15, IFN-γ, TNF-α
CL:
• Localised self healing lesions: epidermal & dermal infiltrate of
histiocytes with amastigotes, lymphocytes & plasma cells
• Non-healing forms: diffuse granuloma with amastigote laden
macrophages (no lymphocytes in DCL)
• Leishmania recidivans: hypersensitive tuberculoid granuloma
with Langhans giant cells with small nos. of lymphocytes and
plasma cells

9. Clinical Features - VL
Most severe form; aka Kala-azar, Dum dum Fever, Black fever
• Incubation period: Avg 2-6 months (10 days to 2 years)
• Symptoms: Fever, fatigue, weakness, loss of appetite &
weight, dragging sensation in left upper abdomen
• Signs: Hepatosplenomegaly, lymphadenopathy, Pallor,
tachycardia & bipedal edema s/o CHF; Hyperpigmentation
• Complications: mainly due to pancytopenia
 Anemia: normochromic normocytic; due to chronic disease,
bone marrow suppression, bleeding, hypersplenism
 Infections: pneumonia, diarrhoea, otitis media, TB
 Bleeding: due to thrombocytopenia & altered hepatic
coagulation factors; epistaxis, gum bleeding, purpura,
menorrhagia
 CHF due to severe anemia

10. Special clinical forms - VL
 Asymptomatic & Sub-clinical Infections: 8.9 : 1 in India
 Post Kala-Azar Dermal Leishmaniasis:
• Chronic rash due to L. donovani in
Asia & East Africa (rarely seen in
HIV/VL co-infection by L. infantum
treated with Sb5+)
• Usually occurs months to years
after treatment of VL with Sb5+
(uncommon with Ampho B)
• Hypopigmented maculae over
papulae to nodules; starts from
face then expands to other parts
(Grades I, II, III)

11. Clinical Features - CL
• aka Delhi boil, Oriental sore, Baghdad boil, Tropical
sore, Aleppo boil, Chiclero’s ulcer, Bouton de Biskra or
Uta
• Sandfly bite → Papule → Painless red nodule → Central
necrosis f/b fall of crust → Ulcer with indurated edge
• Morphology:
Wet sore with healing granuloma: L.mexicana, L.
guyanensis, L. brazilensis, L. major
Dry & squamous lesion: L. tropica, L. peruviana
Flat plaques or hyperkeratotic lesions: Old world CL
Nodular with little inflammation:
L. infantum, L. aethiopica

12. Clinical Features - CL
No systemic symptoms or signs
Lymphangitic dissemination: mainly in L. guyanensis, L.
panamensis, L. braziliensis & L. major
• Lymphangitic cord palpable with small painless nodules
containing parasites
D/D: Sporotrichosis, Nocardiosis, M. marinum, Anthrax,
Tularemia
Evolution: Chronic, spontaneous cure
in months to years with disfiguring
scars (Chiclero’s ulcer in external ear)
and discoloration of skin

13. Special clinical forms - CL
DCL: Severe form of CL;
• Caused by L. aethiopica, L. amazonensis,
L. mexicana; Resistant to therapy
• Multiple, slowly progressive, relapsing
& remitting nodules or plaques without
ulceration, very rich in parasites
• In HIV/Leishmania co-infection:
may be caused by L. braziliensis, L. major,
L. infantum, L. donovani
• D/D: Lepromatous leprosy due to
leonine facies

14. Special clinical forms - CL
Leishmania Recidivans:
• Chronic form due to L. tropica & L. braziliensis
• Usually located on face
• Central healing surrounded by peripheral constantly enlarging
active part (1-2 years after acute lesion)
• Mimics lupus vulgaris; Small no. of parasites present
• Exaggerated cell mediated response

15. Clinical Features - MCL
• Aka Espundia; caused by L. braziliensis & other new world spp
• Primary cutaneous lesion → Variable time of latency →
Mucosal lesions (local spread or metastatic)
• Nasal congestion, epistaxis → Ulceration & perforation of
nasal septum → Tapir nose
• Mucosa of palate (Escomel cross) and lips involved later;
tongue spared. Palatal perforation may occur; Painless
• Laryngeal extension → Dysphonia, metallic cough, obstruction
causing acute dyspnea
• In advanced stage, nose & lips may totally
disappear; nasal & oral cavity connect into
a single hole
• D/D: Paracoccoidomycosis & Malignancy

16. Leishmaniasis & HIV
• 2-5.6% of VL patients may be HIV +ve in India (NVBDCP)
• Re-inforce each other in detrimental manner; impair response
to ART (which can cause PKDL)
• Co-infected patients are highly infectious to sandflies
• Viscerotropic & Dermotropic distinction less valid in co-
infection
• Atypical presentations in patients with CD4 < 200/uL;
cutaneous, mucosal, GI or pulmonary disease; parasite may be
present in lung, pleura, whole of GIT & skin.
• Lower frequency of splenomegaly; Higher frequency & degree
of pancytopenia
• Due to high parasite load, aspirates are more sensitive in
detecting parasites than in immunocompetent.

17. Leishmaniasis & HIV … Contd
• Sensitivity of serological tests like rK39 ↓
• Latex agglutination test: high sensitivity in detecting Ag
in the urine of co-infected patients
• VL is now an AIDS defining illness (WHO); So ART should
be started irrespective of CD4 count.
• 79 to 97% of co-infected patients will relapse (NVBDCP)
• ART should be started 7-10 days after initiating t/t of VL
(NVBDCP)
• Inf Ampho B total dose 40 mg/kg (4 mg/kg on days 1-5,
10, 17, 24, 31, 38) and repeat same dose on relapse

18. Leishmaniasis & HIV … Contd
• Secondary Prophylaxis:
1. Amphotericin B lipid complex 3–5 mg/kg/dose every 3 weeks
for 12 months
2. Liposomal amphotericin B 3–5 mg/kg/dose every 3–4 weeks
3. Pentavalent antimonials 20 mg Sb5+/kg/dose every 3–4
weeks and Pentamidine 4 mg/kg/dose [300 mg for an adult]
every 3–4 weeks
• Prophylaxis could be suspended,
provided that the CD4+ count is
maintained at > 200 cells/μl for > 6 months

19. Diagnosis - VL
Clinical diagnosis: suspect in a patient with
• fever > 2 weeks
• splenomegaly &/or
• weight loss
who lives in or has returned from an endemic area
Routine blood work: isolated or combined presence of
• Anaemia
• Leukopenia
• Thrombocytopenia
• Polyclonal hypergammaglobulinaemia
reinforces clinical suspicion

20. Diagnosis - VL
Parasitological diagnosis: Amastigote form by microscopy of
tissue aspirates → classical confirmatory test
• Sensitivity of aspirates: Spleen (93–99%); Bone marrow (53–86%);
Lymph node (53–65%)
• Stain: Giemsa, Wright’s or Leishman
• Culture of Promastigote:
Novy-MacNeal-Nicolle (NNN),
United States Army Medical Research Unit (USAMRU),
Modified Tobie,
‘Sloppy Evans’
Semi-solid Locke blood–agar.

21. Diagnosis - VL
• Contraindications for Splenic aspiration to be excluded:
(NVBDCP)
1. Hg level is not < 3.0 g/dL
2. No bleeding tendency & not jaundiced
3. Not at an advanced stage of pregnancy
4. Prothrombin time is not > 5 seconds longer than control or
platelet count is not < 40,000/mm3
5. Patient (in case of children) cannot lie still
6. The spleen palpable at least 3 cm below the costal margin
on expiration
7. Vital signs (BP and pulse) are not prohibitive for performing
the procedure

22. Diagnosis - VL
Immunological diagnosis:
• IFAT: Indirect Fluorescence Antibody testing
• ELISA
• Counter-current Immuno-electrophoresis
• Indirect hemagglutination
• Immunoblot techniques
• Easy to use in fields:
o Direct agglutination test
o rK39 ICT
o Dot-ELISA
o Fast agglutination screening test
 2 Limitations:
1. Relapse of VL cannot be diagnosed (Ab persists many years)
2. Asymptomatic individuals harbour Ab in endemic areas

23. rK39 Strip test
• Rapid diagnostic test; Immunochromatographic test
• > 90% specificity and sensitivity
• Results read in 10 minutes in field
• Detects antibodies in blood (even in urine- not validated)
• Test line coated with rK39 antigen – 39-amino acid repeat that
is part of kinesin-related protein in L.chagasi
• Control line region: Chicken anti-protein A.
• The membrane coated with dye conjugate (protein A colloidal
gold conjugate)
• False positives: in Hepatitis and TB.
• False negatives: in HIV
• RDT positive for years after t/t of VL. Not useful in the
diagnosis of relapse.

24. Diagnosis - VL
Antigen-detection tests:
• Latex agglutination test for heat-stable, low-relative
molecular-mass carbohydrate Ag in urine
• Good specificity but low-to-moderate sensitivity in East Africa
and on the Indian subcontinent
• More appropriate for HIV/Leishmania co-infection
DNA PCR (Qualitative or Quantitative):
• In blood, tissue aspirates, urine, buccal swabs
• Most sensitive and specific
• Positive in asymptomatic individuals of endemic areas
• Restricted to referral hospitals and research centres

25. Diagnosis - PKDL
• Diagnosis is mainly clinical; with h/o VL or stay in endemic
area
• Confirmed by finding parasites in skin biopsy or scraping of
skin slit; higher detection when taken from nodular lesions
than papular or macular lesions.
• Skin biopsies: histopathology, immunohistochemistry, culture,
PCR (also in slit skin specimens; more sensitive)
• Serological tests (direct agglutination test, ELISA and the rK39
rapid diagnostic test) usually positive; limited value, positive
result may be due to Ab persisting after a past VL.
• Serology helpful when other diseases (e.g. leprosy) are
considered in the D/D or previous h/o VL is uncertain

26. Diagnosis - CL
Parasitological diagnosis:
• Sample obtained by scraping/ slit skin smear/ FNA/ Bx
• Culture on NNN medium for species identification
DNA PCR: improves diagnostic sensitivity & allows species
identification
Immunological diagnosis:
• Usually no detectable antibody response in L. major or L.
tropica infections

27. Diagnosis - MCL
• Suspect MCL in patients with typical mucosal
lesions and a h/o CL
• Difficult to get samples for parasitological
diagnosis (bleed on contact & difficult to
administer anesthesia)
• Scarce parasites in mucosal lesions (strong local
immune reaction)
• Positive serology (IFAT or ELISA) increase clinical
suspicion.
• DNA PCR – more sensitive tool

28. Treatment of VL - WHO

29. Treatment of VL - WHO

30. Treatment of VL - NVBDCP

31. Treatment of VL in East Africa

32. Treatment of VL – Middle east,
Mediterranean, South America

33. Treatment outcomes in VL

34. Treatment of PKDL - WHO

35. Treatment of PKDL - NVBDCP

36. Treatment outcomes in PKDL

37. Treatment of Old World CL

38. Treatment of Old World CL
• Local Therapy:

39. Treatment of Old World CL

40. Treatment of DCL

41. Treatment of New World CL

42. Treatment of New World CL

43. Treatment of New World CL

44. Treatment of MCL

45. Precautions
• Pentavalent Antimonials: Anorexia, N & V, abdominal pain, metallic
taste, arthralgia, myalgia, acute pancreatitis (in HIV), hepatitis, QT
prolongation (then to VT)
• Amphotericin B: fever with chill and rigor, thrombophlebitis,
myocarditis, hypokalemia, AKI
• Paromomycin: Nephrotoxic, Ototoxic to fetus
• Pentamidine: IDDM, Hypoglycemia, Unexplained shock
• Miltefosine: D & V, hepatotoxicity, renal insufficiency, teratogenic
Contraindications:
• Women during pregnancy & lactation
• Married women of child-bearing age who refuse to give an undertaking
of refraining from pregnancy during the treatment period and
two/three months after completion of treatment
• HIV positive serology
• Infants < 2 years

46. During Pregnancy
• Amphotericin B deoxycholate and lipid
formulations are the best therapeutic options
• Pentavalent antimonials: spontaneous abortion,
preterm deliveries and hepatic encephalopathy
in the mother and vertical transmission (C).
• Paromomycin: Ototoxicity in the fetus is the
main concern.
• Pentamidine is contraindicated during the first
trimester
• Miltefosine is potentially embryotoxic and
teratogenic

47. Prevention & Control
2nd gen VACCINE
• Leish-111f + MPL-SE, reached clinical trials.
• Being evaluated for the immunotherapy of PKDL in the Sudan,
in phase- 1–2 trials in Peru and in a phase-1 trial in India.
Canine leishmaniasis vaccines: in Brazil
Immunochemotherapy and therapeutic vaccines:
• Convit vaccine: autoclaved L. mexicana mixed with BCG
• Mayrink vaccine: killed L. amazonensis vaccine
along with/without chemotherapy

48. Prevention & Control
Sandfly control:
• Destruction of breeding sites
• Insecticide residual spraying (IRS): DDT/Synthetic pyrethroids
• Insecticide treated materials: ITNs
Reservoir Control: Test-and-Treat
Case Detection & Management:
Active and passive surveillance (mainly of PKDL & HIV-VL co-
infection)

49. Recent Publications

50. Recent Publications

51. Road to Kala Azar Elimination