LeishmaniasisLeishmaniasis

Different stages of Haemoflagellates

PromastigotePromastigote Amasitgote Amasitgote TransformationTransformation

Leishmania life cycle

Leishmania life cycle• Amastigotes replicate in

reticuloendothelial cells (mononuclear cells) including;– Monocytes– Macrophages in lymph nodes, spleen, and

lung;– Kupffer cells in sinusoids of liver– Microglial cells in the central nervous

system.– Dendritic cells

Promastigotes reproduce in biting fly (Phlebotomus, Lutzomyia)

Leishmania infects and thrives in macrophages

• Promastigotes attached to CR1 and CR3 receptors on the macrophages

• The parasite invades its host cell passively by phagocytosis (parasitophorous vacuole)

Ovoid small intracellular parasites in a bone marrow aspirate. The typical rod shaped kinetoplast is seen besides

the nucleus.(Giemsa stain).

Leishmania sp.

amastigote stage

Leishmaniasis vectors• There are over 600 species of sand flies divided into

five genera. More than 30 species are proved vectors.

• Phlebotomus in the Old-world and Lutzomia in the New world are vectors of human leishmaniasis.

Procyclics and Metacyclic Promastigotes

• Amastigotes are released by digestion, transform into procyclic promastigotes and attach to the midgut epithelium

• Attached promastigotes divide rapidly

• Metacyclic (infective) promastigotes cease replication, detach and pass forward into the pharynx from where they are regurgitated into the bite site

(attached)

(detached)

• subgenus Leishmania: develops in the sand fly’s midgut and foregut (suprapylorian)– Both Old world and New world visceral and cutaneous species

• subgenus Viannia: develops in the hindgut and midgut (peripylarian).– L. braziliensis complex, L. guyanensis complex.

Two subgenera of Leishmania genus

Importance Importance • Leishmaniasis is a parasitic disease transmitted by the bite of sand flies.• In at least 88 countries.• 350 million people at risk. • 12 million people are affected by leishmaniasis. • 1.5-2 million new cases of cutaneous leishmaniasis estimated to occur

annually.• 500 000 new cases of VL which occur annually.• 90% of CL cases were from Afghanistan, Algeria, Brazil, Iran, Peru,

Saudi Arabia and Syria.• 90% of VL cases were from Bangladesh, India, Nepal, Sudan and

Brazil.• 90% of mucocutaneous leishmaniasis occurs in Bolivia, Brazil and Peru.

Endemic areas for leishmaniasisEndemic areas for leishmaniasis

1. Cutaneous leishmaniasis (CL)

2. Visceral leishmaniasis (VL), (kala-azar) is the most severe form of the disease. Mortality rate 75-95%

3. Mucocutaneous leishmaniasis (MCL), or espundia, disfiguring, destruction of mucous membranes of the nose, mouth and throat cavities. Reconstructive surgery of deformities is an important part of therapy

The disease main forms

Cutaneous LeishmaniasisCutaneous Leishmaniasis

Cutaneous LeishmaniasisSkin ulcers on the exposed parts of the body such

as the face, arms and legs.

Old World: L. major, L. tropica, L. aethiopica (DCL)

New World: L. mexicana, L. pifanoi, L. amazonensis, L. venezuelensis, L. granhami,

Cutaneous LeishmaniasisCutaneous Leishmaniasis• Characterized by one or

more papules, nodules and sores on the skin

• Sore like a volcano with a raised edge and central crater.

• Two figures including urban (dry) and rural (wet) forms.

• Sores are usually painless but can become painful if secondarily infected

• chronic but self-limiting

• Infection remains restricted to the initial site of infection (the bite site)

Leishmania tropica

• Anthroponotic Cutaneous Leishmaniasis (ACL)

• Definitive Host: Humans• (occasionally dog?)

• Intermediate Host: Phlebotomus sand flies

– Main vector and also in Iran: P. Sergenti

• Dry or urban C.L.• Face> hand, leg and…• Incubation period: 2-8 months(usually 2-3

months)• Lesion persist for several months (more

than one year) then person is immune

Leishmania tropica

• Sores don’t heal very quickly– Often mistaken for

leprosy or tuberculosis

• First sign: small papule nodule dry sore

• Itching• Scar (if not treated)

• Rarely can cause visceral (viscerotropic) and diffuse cutaneous infections.

Leishmania major

• Wet or rural form• Definitive Host: rodents (Rodentia: Gerbillidae) as reservoir

host, Humans• In Iran: Rhombomys opimus, Meriones libycus,

M.hurrianae, Tatera indica • Zoonotic Cutaneous leishmaniasis (ZCL)• Intermediate Host: Phlebotomus sand flies. In Iran: P. papatasi.• Incubation period: some weeks to 3 months ( usually 2

weeks)• First sign: small papule nodule Wet sore (exudates)

• Itching• Scar (if not treated)• Disease period is usually short (2-8 months)• More in hands and feet

Diffuse cutaneous leishmaniasis

Recidivans leishmaniasis

Sporotrichoid form

Leprosy

• It is primarily a granulomatous disease of the peripheral nerves and mucosa of the upper respiratory tract.

• Skin lesions are the primary external sign.

• Left untreated, leprosy can be progressive, causing permanent damage to the skin, nerves, limbs and eyes.

Lupus volgaris (a sort of cutaneus TB)

• Persistent and progressive form of cutaneous TB.

• Small sharply defined reddish-brown lesions with a gelatinous consistency (called apple-jelly nodules).

• Lesions persist for years, leading to disfigurement and sometimes skin cancer

New World cutaneous leishmaniasis

• L. mexicana (Chicleros Ulcer)– External ear

• L. pifanoi• L. amazonensis

– main cause of DCL

– All four leishmaniasis forms

• L. (V.) peruviana (Uta)• L. granhami• L. venezuelensis

Visceral LeishmaniasisVisceral Leishmaniasis

• Systemic infection of reticulo-entdothelial cells (mostly macrophages) throughout multiple internal organs and the blood

Kala-azar , Visceral Leishmaniasis

• L. donovani complex

– L. donovani (Asia, East Africa)

• Strain archibaldi

– L. infantum (Medditeranian basin and Middle East, in children)

– L. chagasi ( South & Central America)

• L. tropica (viscerotropic)

• L. amazonensis

• Weeks to months (2-6 months) incubation period

L. infantum, zoonosis,

L. donovani ,(archibaldi), zoonosis,

L. donovani, anthroponotic

L. chagasi, zoonotic

The most sever leishmaniasis form

• Mortality of untreated disease 75-95%

• Prolonged fever (usually dromedary but also continuous, reminant, interminnent)

• Splenomegaly, hepatomeglay,

• Weight loss (cachexia),

• Progressive anemia, pancytopenia,

• Hypergammaglobulinemia and hypoalbominemia

• Skin darkness ( around mounth, forehead, temple)• Lymphadenopathy may be present• Elevated liver enzymes, Nausea, vomiting.• Pancytopenia: normochrome & normocytic anemia,

trombocytopenia, but lymphocytosis

• Visceral leishmaniasis should be considered in every case with chronic fever returning from an endemic area.

• Malaria, tropical splenomegaly, schistosomiasis, cirrhosis, portal hypertension, trypanosomiasis, milliary tuberculosis, brucellosis, typhoid fever, bacterial endocarditis, histoplasmosis, malnutrition, lymphoma, and leukemia (Singh, 2006).

• Apart from fly biting, other routes including; Placental, blood transfusion, mechanical, sexual routes.

Visceral LeishmaniasisVisceral Leishmaniasis

Viscerotropic leishmaniasis

• L. tropica

• Oligoparasitic leishmaniasis

• Fever, fatigue, digestive tract problems

• First in desert storm operation in Iraq among American soldiers

• In Iran: One case from AIDS patient (Tehran), other case (Shiraz)

Epidemiology of visceral leishmaniasis in Iran

Mediterranean Kala-azar:

- Causative agent is L. infantum

- Reservior host: involving canine such as Dog, Jackal, fox, wolf and other wild carnivorouses.

- The probable main vector in Iran is Phlebotomus major (Fars), other vectors are P.keshishiani (Fars), P. Perfiliewi (Dashte-Moghan),P. kandelakii (Meshkinshahr).

- Age distribution: the disease mainly occurs in children from 1 to 4 years of age.

Immunity

• TH1:– Macrophage produces IL12– IL12 promote TH0 to TH1– TH1 produces INF gama, IL2, TNF– Healing and resistance to disease

• TH2:– TH2 produces IL4, IL5, IL6, IL10, IL13– Sensitivity to disease

DiagnosisDiagnosis

• 1. Clinical Diagnosis:

– Patient history ( endemic region or travel),– Signs & symptoms

• Sores that will not heal, have to be referred for evaluation.

• Individuals with fevers, weight loss, gastrointestinal complaints, anemia, hypergammaglobulinemia, abnormal liver tests should be referred for evaluation

1. 1. Cutaneous LeishmaniasisCutaneous Leishmaniasis

• Tissue sample (scraping, aspirate or punch biopsy) for smear and culture

• Take scrapings from the sore, put on slides, stain with Wright’s or Giemsa’s stains, and look for amastigotes.

• Culture (NNN & LIT, Evans, RPMI 1640),

• Laboratory animals inoculation (Souri andBalb/c mouse) only for L. major (no growth in L. tropica)

Laboratory Diagnosis of leishmaniasis :

Cutaneous Leishmaniasis (con.)Cutaneous Leishmaniasis (con.)

• Leishmania skin test (Montenegro test)

– 0.1 ml (1,000,000 killed L. major promastigote), intradermal,

– 5mm< induration after 48-72h, DTH

• Isoenzyme Isoenzyme profiles - Zymodemesprofiles - Zymodemes

• No serological approaches No serological approaches usually but monoclonal usually but monoclonal antibodies can be used.antibodies can be used.

• DNA hybridisation - PCRDNA hybridisation - PCR

2. 2. Visceral LeishmaniasisVisceral Leishmaniasis

• Finding Leishmania on biopsy of bone marrow (iliac, sternum, tibia (54-86% sensitivity)), liver (60%), enlarged lymph node (64%), or spleen (98%).

• Culture (NNN, Evans, LIT)

• Laboratory animals IP inoculation (Golden hamester)

• No LST for VL and PKDL diagnosis (Yes for VL and CL epidemiology and MCL and Lupoid diagnosis)

Visceral Leishmaniasis (con.)Visceral Leishmaniasis (con.)

• Serologic tests:

– Antibody detection: DAT (sen 91-100%, spe 72-95%) IFA (sen 55-70, spe 70-89) ELISA,( sen 80-100% spe 84-95) Dipstick test (rk39, recombinant antigen 39kd, sen 67-100% spe 88-100%)

• DAT is easy, inexpensive, and with high specificity and sensitivity (most usage in Iran)

– Antigen detection: KAtex (5-20 kd glycoprotein, membranous antigen, easy, field applicable, sen 68-100%, spe 100%, positive only in acute disease, useful for HIV/VL )

• Formel gel, (Based on hyperimmunoglobulinemia)Formel gel, (Based on hyperimmunoglobulinemia)

– Multiple myeloma, Schistosomiasis Multiple myeloma, Schistosomiasis

• Isoenzyme profiles - ZymodemesIsoenzyme profiles - Zymodemes

• Monoclonal antibodiesMonoclonal antibodies

• DNA hybridisation – PCR (Schizodem)DNA hybridisation – PCR (Schizodem)

Cutaneous and mucocutaneous treatmentCutaneous and mucocutaneous treatment

• Antimony components : Meglumine antimoniate (Glucantime) and Sodium stibogluconate (Pentostam) are drugs of choice.– 20 mg/kg/d IV or IM for 20d

- Pentamidine, Paromomycin are alternative drugs for CL

- Amphotricine B for antimony resistant MCL

• Fluconazole may decrease healing time

Visceral leishmaniasis treatment Visceral leishmaniasis treatment • Pentostam or Glucantime 20 mg /kg/d IV or IM for 28d

• Amphotricin B: 0.5-1 mg/kg IV daily 15-20d

• Liposomal Amphotricin B (Ambisome): 3 mg/kg/d IV on days 1-5, day 14 and day 21

– Low toxicity and high stability, better delivery

• Alternative: Pentamidine (4mg/kg three times weekly, between 5-25 weeks ), Parmomycine

Visceral leishmaniasis treatment (con.)Visceral leishmaniasis treatment (con.)

• Miltefosine (Impavido) (2.5 mg/kg /d p.o. for 28 d)– It was developed for cancer therapy at first– The only oral drug– safer and more tolerable drug (less toxicity for

bone marrow and haematopoietic progenitor cells) – teratogenic

Leishmaniasis control• Vector control

– insecticides

– insecticide impregnated bed nets (IIB)

• Case finding treatment

• Aniaml reservoir control

– Treatment or killing of seropositive dogs

– Rodent killing

•  

• Decrease of susceptibility: Childhood age, malnutrition and Immunosuppression are susceptibility factors for VL.

– eliminating of childhood malnutrition

– try to produce an efficient

vaccine 

• 0.1 ml in each week for 12 weeks

لیشمانیوز ضایعات از برداری نمونه روشپوستی

• ( لبه پوستی ضایعات متورم و ملتهب های کناره. ) دارد را آمستیگوت میزان بیشترین زخم خارجی

آمستیگوت • یافتن شانس بیشتر بافت چه هربیشتر

الکل • و پنبه با زخم محل کردن توجه% 70تمیز با ،ضایعه روی بر قارچی و باکتریایی عفونت به

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شود.سمت • به خراشهایی شده شکافته محل عمق از

داده نمونه برداشت جهت ضایعه مرکز و سطح. شود می

پوستی ادامه لیشمانیوز ضایعات از برداری نمونه روش

شده • برداشت مواد از و خارج را اسکالپل تیغه اسمیر 3نوکبیمار اسم یا نمونه الماسشماره قلم با و شود می گرفته

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شیئی • عدسی با سپس 10مشاهده سپس 40و ( 100و ایمرسیون ) روغن باالمل بدون ومستقیما

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مجدد

ایزوالسیون

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مایع فاز عنوان

کشتادامه

خون • و استخوان مغز آسپیراسیون پوستی، بیوپسی نمونهدار 2• شیب سطح ترین پایین عمق در متر میلیدر • سانتیگراد 25-22اینکوباسیون درجه•. شد خواهد یافت و کرده رشد مایع فاز در انگل

• : هستند انگل رشد مانع قارچ و باکتری کردن کار .استریل• : تا و یکبار ای هفته انگل جستجوی جهت کشت بررسی

.) است ) بودن منفی معادل رشد عدم یکماه–. دارد نیاز را بیشتری زمان باشد رشد دیر انگل اگر