leishmaniasis n.
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Leishmaniasis. Different stages of Haemoflagellates. Leishmania life cycle. Promastigote Amasitgote Transformation. Leishmania life cycle. Amastigotes replicate in reticuloendothelial cells (mononuclear cells) including; Monocytes Macrophages in lymph nodes, spleen, and lung;

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Leishmania life cycle



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.
    • Dendriticcells

Promastigotes reproduce in biting fly (Phlebotomus, Lutzomyia)

leishmania infects and thrives in macrophages
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)

Leishmania sp.

amastigote stage

  • Ovoid smallintracellular parasites in a bone marrow aspirate. The typical rod shaped kinetoplast is seen besides the nucleus.(Giemsa stain).
leishmaniasis vectors
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
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) promastigotescease replication, detach and pass forward into the pharynx from where they are regurgitated into the bite site



two subgenera of leishmania genus
Two subgenera of Leishmania genus
  • 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.
  • 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.

The disease main forms

  • 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
cutaneous leishmaniasis1
Cutaneous Leishmaniasis

Skin 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 leishmaniasis2
Cutaneous 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
leishmania tropica
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 tropica1
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)
leishmania major
Leishmania major
  • Wet or rural form
  • Definitive Host: rodents (Rodentia: Gerbillidae) as reservoir host, Humans
  • In Iran: Rhombomysopimus, Merioneslibycus, M.hurrianae, Tateraindica
  • 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

Sporotrichoid form

Diffuse cutaneous leishmaniasis

Recidivans leishmaniasis

  • 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
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
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
Systemic infection of reticulo-entdothelial cells (mostly macrophages) throughout multiple internal organs and the blood
kala azar visceral leishmaniasis
Kala-azar , Visceral Leishmaniasis
  • L. donovanicomplex
    • 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. donovani, anthroponotic

L. infantum, zoonosis,

L. donovani ,(archibaldi), zoonosis,

L. chagasi, zoonotic

the most sever leishmaniasis form
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 leishmaniasis1
Visceral Leishmaniasis
  • 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.
viscerotropic 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
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.

  • 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
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

Laboratory Diagnosis of leishmaniasis :

1. Cutaneous 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 (SouriandBalb/c mouse) only for L. major (no growth in L. tropica)
cutaneous leishmaniasis con
Cutaneous Leishmaniasis (con.)
  • Leishmania skin test (Montenegro test)
    • 0.1 ml (1,000,000 killed L. majorpromastigote), intradermal,
    • 5mm< induration after 48-72h, DTH
  • Isoenzymeprofiles - Zymodemes
  • No serological approaches usually but monoclonal antibodies can be used.
  • DNA hybridisation - PCR
2. Visceral Leishmaniasis
  • Finding Leishmaniaon 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)
    • Multiple myeloma, Schistosomiasis
  • Isoenzyme profiles - Zymodemes
  • Monoclonal antibodies
  • DNA hybridisation – PCR (Schizodem)
cutaneous and mucocutaneous treatment
Cutaneous 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
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
روش نمونه برداری از ضایعات لیشمانیوز پوستی
  • کناره های ملتهب و متورم ضایعات پوستی(لبه خارجی زخم) بیشترین میزان آمستیگوت را دارد.
  • هر چه بافت بیشتر شانس یافتن آمستیگوت بیشتر
  • تمیز کردن محل زخم با پنبه و الکل 70%، با توجه به عفونت باکتریایی و قارچی بر روی ضایعه سالک
  • خشک شدن الکل قبل از نمونه برداری
  • محل نمونه برداری توسط انگشت شصت و سبابه محکم گرفته شود.
  • توسط تیغه اسکالپل نوک باریک و یا لانست استریل شکافی به عمق یک میلی متر در ناحیه ای که توسط دو انگشت گرفته شده ایجاد می شود.
  • از عمق محل شکافته شده خراشهایی به سمت سطح و مرکز ضایعه جهت برداشت نمونه داده می شود.
ادامه روش نمونه برداری از ضایعات لیشمانیوز پوستی
  • نوک تیغه اسکالپل را خارج و از مواد برداشت شده 3 اسمیر گرفته می شود و با قلم الماس شماره نمونه یا اسم بیمار بر روی لام شیشه ای ثبت شود.
  • در صورت نیاز به کشت، در کنار شعله نمونه ( بر روی تیغه اسکالپل) به درون محیط کشت دایفازیک منتقل می شود.
رنگ آمیزی گیمسا
  • محلول تجارتی غلیظ (و متفاوت)
    • پس از رقیق سازی بتواند گلبول سفید را به خوبی رنگ کند.
  • اسلاید حاوی نمونه بدون شعله ودر دمای اتاق خشک شوند.
  • متانول Methanol 70%
  • متانول خشک شود (اصطلاحا بپرد)
  • بسته به دستور تولید کننده مثلا 1 به 30 یا 1 به 50 رنگ رقیق شود.
    • آبی که جهت رقیق کردن استفاده می شود جهت7.2 pH تنظیم شده باشد.
  • بسته به رقت و نوع آن مدت رنگ آمیزی 20-30 دقیقه است که به طور تجربی بدست می آید.
  • مشاهده با عدسی شیئی 10 و سپس 40 و سپس 100 (با روغن ایمرسیون) ومستقیما بدون لامل
  • حداقل جستجوی 30 شان و یافتن ماکروفاژها در هر کدام از 3 لام
    • نبود ماکروفاژ و فراوانی گلبول قرمز نشانه نمونه نامناسب inadequate)) و ضرورت نمونه گیری مجدد
ایزولاسیونکشت در محیط NNN
  • Novy, Macneal, Nicolle
  • آگار 14 گرم
  • NaCl 6 گرم
  • آب مقطر cc 900
  • حرارت (جوشاندن) تا شفاف شدن ( ولی احتیاط جهت سر ریز نشدن)
  • اتو کلاو و سپس سرد شدن تا حرارت 50-60 درجه
  • اضافه کردن گلوکز 30گرم در صد به میزان 5 درصد
  • اضافه کردن خون خرگوش دفیبرینه 10-12 درصد حجم محلول
  • به صورت گرم، تقسیم در لوله های استریل درپیچ دار (3 میلی لیتر)، قرار دادن به شکل slant
  • انتقال به یخچال پس از بستن آگار
  • در هنگام استفاده، اضافه کردن 0.5 تا 1 سی سی PBS یا RPMI به عنوان فاز مایع
ادامه کشت
  • نمونه بیوپسی پوستی، آسپیراسیون مغز استخوان و خون
  • 2 میلی متر در عمق پایین ترین سطح شیب دار
  • اینکوباسیون در 22-25 درجه سانتیگراد
  • انگل در فاز مایع رشد کرده و یافت خواهد شد.
  • استریل کار کردن: باکتری و قارچ مانع رشد انگل هستند.
  • بررسی کشت جهت جستجوی انگل: هفته ای یکبار و تا یکماه ( عدم رشد معادل منفی بودن است).
    • اگر انگل دیر رشد باشد زمان بیشتری را نیاز دارد.