8/17/2021

Leishmania 1
Dr. Samar Habib, MD, PhD
Department of Medical Parasitology, Mansoura Faculty of Medicine

Contents of the lecture

 Taxonomic classification
 Morphology and life cycle
 Clinical syndromes of CL
 Old world CL
 Diagnosis of old world CL
 Treatment of of old world CL
 Vaccination
 New world CL

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Taxonomic classification
Phylum: Euglenozoa
Order: Kinetoplasida

Common ancestors
Genetic exchange

Morphology and life cycle

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Clinical syndromes of cutaneous leishmaniasis

1. localized ulcerative or nodular cutaneous leishmaniasis

(CL)
2. Mucosal leishmaniasis (ML)…. Jungles of Amazonia
3. Leishmaniasis recidivans (LR)…… Middle East
4. Diffuse or disseminated cutaneous leishmaniasis (DCL)
5. Post-kala azar dermal leishmaniasis (PKDL)
6. Immunocompromised patients may have atypical
presentations

Old world CL

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Old world (Eastern)CL

 Leishmania major, L. tropica, L. aethiopica

 Oriental sore, Baghdad boil, Delhi boil, Biskra button, and Aleppo
evil.
 Various surface molecules on the promastigote, such as gp63 and
lipophosphoglycans (LPG), help bind the parasite to the host
macrophage receptors, thus allowing it to be phagocytized.
 The amastigotes remain confined to the skin
 Lesions usually occur on exposed parts of the body such as the
face, hands, feet, arms, and legs.

Leishmaniavirus

 Is a double-stranded RNA virus that persistently infects some strains

of Leishmania.
 There is a possibility that this virus may be able to alter the parasite
phenotype and may affect disease pathogenesis.
 The virus has been detected in cultured parasites and has also been
detected in human biopsy samples prior to manipulation in culture

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Clinical disease of old world CL

 The incubation period of CL is usually 2 to 8
weeks but can be several years.
 The first sign of cutaneous disease is a lesion
(generally a firm, painless papule) at the bite
site, multiple lesions of L. major or disfiguring
facial lesions of L. tropica may be
psychologically or physically devastating.
 All lesions on a patient have a similar
appearance and progress at the same speed.
 The original lesion may remain as a flattened
plaque or may progress, with the surface
becoming covered with fine, papery scales.

 These scales are dry at first but later

become moist and adherent,
covering a shallow ulcer.
 As the ulcer enlarges, it produces
exudate and may develop a crust.
 The edge of the lesion is usually
raised.
 Depending on the species, satellite
lesions are common and merge with
the original lesion

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Leishmania major (rural disease)

 Multiple lesions are present and are accompanied by
marked inflammation and crusting.
 The center of the lesion tends to be necrotic and
exudative, forming a loose crust.
 The lesions tend to mature and heal relatively quickly,
often lasting only a few months.
 Lymphatic spread may occur in L. major infections, with
subcutaneous nodules in a linear distribution and
regional lymphadenopathy
 May result in severe scarring.

Leishmania tropica (urban disease)

 Occurs singly or two at a time

 Develop more slowly than those caused by L. major, and can take a
year or more to heal.
 They tend to be more swollen and less necrotic, with less exudate
and a thicker crust.

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Leishmania tropica (Leishmaniasis Recidivans)

 LR is generally associated with L. tropica

 Small, non-ulcerating lesions begin to appear, mainly on the margins of healed
lesions, and continue to expand the limits of the original scar
 Disfigurement due to extensive scarring can develop.
 May be associated with a strong cell-mediated immune response and tends to
occur in Iran and Iraq.
 Although there is a strong immune response, not all amastigotes are eliminated,
and the infection can last for 40 years.
 Parasites may be very difficult to demonstrate; however, culture or animal
inoculation has been successful. The leishmanin skin test (LST) (Montenegro test)
is strongly positive in these patients.

L. aethiopica (Cutaneous Leishmaniasis)

 The least inflamed but more swollen.

 Scaling and exfoliation of the dermis occur.
 These lesions tend to remain for years prior to complete healing.

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L. aethiopica (Diffuse Cutaneous Leishmaniasis-

DCL).
 Anergic form of CL in which neither
humoral nor cell-mediated immune
responses are functional
 DCL is caused by L. aethiopica and is seen
in Ethiopia and Kenya
 Thickened skin in plaques, papules, or
multiple nodules.
 Confused with leprosy.
 Although lesions are usually seen on the
face, nose, limbs, and buttocks, they can
occur over the entire body

 The nodules are often described as soft and fleshy, while those of
leprosy are generally more indurated.
 No visceral lesions.
 Treatment is difficult, and the infection may last for the life of the
patient.
 The LST remains negative unless treatment is effective and recovery
occurs.

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Old World cutaneous leishmaniasis, subgenus Leishmania: vectors, hosts, and

lesions

Diagnosis of Cutaneous Leishmaniasis

 In endemic areas, the diagnosis may be made on clinical grounds,

However, in other areas of the world, the condition may not be
recognized.
 Definitive diagnosis depends on demonstrating the amastigotes in
tissue specimens or the promastigotes in culture

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Specimen collection
 If the patient has multiple lesions, specimens should be collected from the more
recent or active lesions.
 Thoroughly clean with 70% alcohol, and remove necrotic debris to prevent the risk of
bacterial and/or fungal contamination of the specimen, which is mandatory if cultures
are to be used for isolation and growth of the parasites; excess bacterial or fungal
contamination prevents development of the promastigote forms.
 Specimen should be taken from the advancing margin of the lesion; the central portion
of the ulcer contains nothing but necrotic debris.
 Local anesthesia…If specimens are collected by aspiration, scraping, or biopsy
 The specimen of choice would be a collection of several punch biopsy specimens taken
from the most active lesion areas.
 Biopsy specimens can be divided and used for cultures and touch preparations; some
material should always be saved and submitted for routine histology.

 Impression smears: After any excess blood is cleaned off, a

horizontal cut is made through the biopsy core and the cut surface
is gently touched to glass slides; multiple slides should be prepared.
 The amastigotes are more difficult to identify in tissue sections than
in touch preparations; they appear smaller and may be cut at
different angles.
 Staining Giemsa intracytoplasmic kinetoplast confirms the
identification

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 Culture  tissue mincing is recommended to release the parasites/

not grinding
 M-199 medium, Novy-MacNeal- Nicolle medium, and Schneider’s
Drosophila medium supplemented with 30% fetal bovine serum
 Both the control and patient cultures should be examined twice a
week for the first 2 weeks and once a week for up to 4 weeks before
they are reported as negative.
 Promastigotes can be detected microscopically in wet mounts taken
from centrifuged culture fluid. This material can also be stained with
Giemsa stain to facilitate observation at a higher magnification.

 Animal Inoculation. Animal inoculation can be helpful when only a

small number of organisms are present…. no longer considered
practical.
 Golden hamsters are inoculated intranasally, and the major
argument against this approach is that it may take several months
before the animal gives positive responses.

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 Skin Test. The leishmanin skin test (LST) (Montenegro test) is

useful for epidemiologic surveys to detect groups at risk for
infection.
 The test is not species specific and must be interpreted with
caution, particularly since some patients may have more
than one type of leishmaniasis.
 The LST involves a delayed hypersensitivity reaction to a
suspension of killed promastigotes.
 The reaction is read at 48 and 72 h after injection. Positive
reactions are generally seen in patients with cutaneous
lesions; however, those with DCL give negative reactions

 Serologic Tests. Although available for epidemiology and/ or research studies,

serologic tests are not very useful for the diagnosis of CL.
 Reagent availability is usually limited to areas of endemic infection.
 Antibody detection is variable in patients with CL; if antibody is present, titers are
usually low, and cross-reactions have been seen in patients with leprosy.
 Patients infected with the viscerotropic form of L. tropica had low or
undetectable antibody titers.

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Treatment of old world CL

 Most Old World CL lesions are self-healing over a period of a few

months to several years.
 This healing process confers immunity to individuals living within
the immediate area of endemic infection; therefore, treatment is
not recommended unless disfiguring scarring is a possibility.
 Chemotherapy should be given to patients with lesions on the face,
specially if the lesions are multiple or large

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 Cutaneous lesions, whether treated or not, should always be cleaned and

covered to prevent secondary infections and vector access to the lesion.
 Pentavalent antimonials (sodium stibogluconate [Pentostam]) are usually
effective
 Miltefosine is another option, with paromomycin or pentamidine being
alternatives. Toxicity does not usually present a problem.
 The lesions of LR respond to Pentostam at doses used for treatment of VL, and
local heat therapy and intralesional injections are commonly used in the Middle
East.
 In treating DCL, Pentamidine, seems to be a good compromise between efficacy
and toxicity.

 Other more local treatment options have included cryotherapy, heat, and surgical
excision of lesions.
 Organisms within the L. tropica group do not survive in temperatures above 37°C, and
local heat therapy has proven to be effective.
 The temperature within the lesion should reach 40 to 42°C for 12 h at a time….infrared
heat lamp.
 The use of hot water in baths or saunas has been helpful in cases of DCL, particularly
since the response to chemotherapy is poor.
 The administration of Pentostam as direct injection into the lesion has also been used.
The dose is divided into four parts and injected into four different quadrants of the
lesion.

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Vaccination
 In areas of Iran, Iraq, Jordan, and Russia where CL is endemic, vaccination has been used
for many years.
 Exudate from a naturally acquired lesion can be inoculated onto a hidden area of the body
of a nonimmune person.
 Another approach is to use live, attenuated L. major promastigotes, which are inoculated
intracutaneously.
 The infection is allowed to develop and is usually limited to a single lesion; self-cure
proceeds as in a natural infection.
 The immunized individual will become a carrier and source of infection until the lesion
heals
 A very small percentage of those vaccinated will develop lesions requiring treatment.
 Lifelong immunity to the homologous species develops and is comparable to that
following a natural infection.

New world CL

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New world (Western)CL

 Extends from southern Texas and Arizona to Latin and South
America and the Caribbean islands
 Wide range of disease presentation, and separation based on
clinical signs and symptoms is not possible
 Organisms in this group belong to the subgenera Leishmania and
Viannia
 Within the subgenus Leishmania is the L. mexicana complex and
within the subgenus Viannia are the L. braziliensis complex and the
Leishmania guyanensis complex
 Taxonomy will continue to change as molecular methods reveal
genetic differences and similarities among the various genera and
species.

 Common names: uta (Peru), chiclero ulcer or bay sore (Mexico),

and forest yaws (Guyana)

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Life cycle and morphology

 The same as old world CL
 Sandfly vector is in the genus Lutzomyia
 The genus Leishmania is divided into its two subgenera, Leishmania
and Viannia, depending on the location of the development of the
promastigote within the sand fly.
 Promastigotes from the subgenus Viannia develop in the midgut
and hindgut of sand flies, while promastigotes from the subgenus
Leishmania develop in the anterior portion of the sand fly
alimentary tract

Clinical Disease: cutaneous leishmaniasis

(CL)
 Cutaneous Leishmaniasis. The lesions of New World CL are very
similar to those seen with Old World cutaneous disease.
 Weeks to months after infection, an erythematous, often pruritic
papule develops at the bite site. This papule may become scaly
and enlarge, developing a central ulcer surrounded by a raised
margin.
 Disease progression at this point will vary depending on the
species involved. Lesions may be single or multiple and usually
occur on exposed areas of the body.
 Many lesions may self-heal within 6 months; however, lesions on
the ear occur in 40% of the patients and are chronic, lasting
many years. Ulcerations on the ears may be quite destructive but
cause the patient few problems.

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 L. Mexicana lesions are usually single, self-limiting

cutaneous papules, nodules, or ulcers that are painless
and are found on the face and ears.
 Other infections that are usually associated with single
cutaneous lesions include those due to Leishmania
colombiensis, Leishmania amazonensis, Leishmania
garnhami, Leishmania lainsoni, Leishmania peruviana,
and Leishmania venezuelensis.
 Multiple lesions caused by spread along lymphatics are
common with L. guyanensis infections, and subcutaneous
lymphatic nodules can be seen with Leishmania
panamensis

L. major
L. guyanensis
L. panamensis

New World cutaneous leishmaniasis subgenus Leishmania,

complex Leishmania mexicana

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Clinical Disease: Mucocutaneous

leishmaniasis (ML)
 ML is most common in southern Brazil, but cases have been reported throughout
the Americas.
 Cutaneous lesions caused by L. braziliensis usually require a longer period for
self-cure.
 Approximately 1 to 3% of patients infected with L. braziliensis develop metastatic
spread of the organisms to the nasal, pharyngeal, and buccal mucosa (ML).
 ML, also known as espundia, may occur months to years after the initial
cutaneous lesion has healed partially or completely.
 More frequent in males than females, and the patients are usually 10 to 30 years
of age.

 ML may begin with erythema and edema of the involved mucosal tissues,
along with nasal inflammation, stuffiness, difficulty in breathing through
the nose, and occasional bleeding.
 Following the early symptoms, ulcerations covered with a mucopurulent
exudate may develop. There is severe erosion and destruction of the soft
tissues and cartilage, leading to loss of the lips, soft parts of the nose, and
soft palate.
 The lesions are chronic and progressive and can lead to death due to
secondary infection or aspiration pneumonia.
 In the non-ulcerative form, there may be hypertrophy of the tissues of the
upper lip and nose, leading to the development of the “tapir nose.”
 Although this disease can be extremely disfiguring, the mortality rate is low.

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New World cutaneous leishmaniasis subgenus Leishmania, complex Leishmania braziliensis

New World cutaneous leishmaniasis, subgenus Viannia, complex Leishmania guyanensis

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Clinical disease: Diffuse or Disseminated

Cutaneous Leishmaniasis (DCL)
 Anergic form of CL in which neither humoral nor cell-mediated immune
responses are functional
 Most frequently in Venezuela, Brazil, and Mexico.
 Individuals infected with Lesihmania pifanoi and 30% of those infected
with L. amazonensis are likely to develop DCL
 The parasites are found in the skin, and the disease is characterized by
the formation of thickened skin in plaques, papules, or multiple nodules.

 Although lesions are usually seen on the

face, nose, limbs, and buttocks, they can
occur over the entire body.
 The nodules are often described as soft
and fleshy, while those of leprosy are
generally more indurated.
 No visceral lesions
 Treatment is difficult, and the infection
may last for the life of the patient.
 The LST remains negative unless
treatment is effective and recovery
occurs

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DCL: Complex Leishmania mexicana

Diagnosis

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The same as old world CL

 Specimen collection
 Staining
 Culture
 Animal inoculation
 Skin test
 Serologic test
 PCR is a valuable tool for the diagnosis of leishmaniasis on a routine basis and
can provide valuable epidemiologic information in areas of endemic infection.

Treatment of new world CL

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 In contrast to Old World CL, systemic chemotherapy is recommended for New

World CL.
 Cases with multiple lesions; the long-term, chronic nature of some infections;
and the tendency for disease progression and mucosal involvement in the
absence of chemotherapy.
 Recombinant human granulocyte macrophage colony-stimulating factor (GM-
CSF) coupled with stibogluconate (in studies)
 Allopurinol and stibogluconate is good for CL but not ML (in studies)

 The most promising drug found so far is miltefosine, which has undergone
experimental and clinical trials and is 94 to 97% effective.
 However, the drug is contraindicated during pregnancy and may cause severe
gastrointestinal side effects. Also, the cost is another limiting factor.
 In Mexico, treatment with localized current field radiofrequency device to
generate controlled heat appears to be effective. Lesions are first anesthetized
with 1% lidocaine hydrochloride and moistened with normal saline solution.
Treatment consists of a single application of heat that produced 50°C for 30 s.

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Vaccines

 Current control of leishmaniasis is based on chemotherapeutic treatments which

are expensive, toxic, and associated with high relapse and resistance rates.
 Vaccination remains the best hope for control of all forms of the disease.
 The development of a safe, effective, and affordable anti-leishmanial vaccine is a
critical public health priority.
 Extensive evidence from studies in animal models indicates that solid protection
can be achieved by immunization with defined subunit vaccines or live-
attenuated strains of Leishmania.
 To date, no vaccine is available despite substantial efforts by many laboratories
 Although not currently available, research and potential trials continue to be
undertaken for vaccine development.

References

 Diagnostic medical parasitology, Garcia 2016

 CDC

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