NEGLECTED TROPICAL DISEASES

LEISHMAIASES

IN THE NAME OF ALLAH

LEISHMAIASES
INTRODUCTION :
Leishmaniasis is one of the most important vector-borne diseases of humans. This
parasitic disease can be caused by many species of Leishmania, most of which are
zoonotic. In humans, different species of the parasite are associated with different forms
of the disease. Many Leishmania spp . cause skin ulcers and nodules. A few of these
organisms can also affect the mucous membranes, and may cause disfiguring lesions of
the nose. Other species
damage the internal organs and cause human visceral leishmaniasis, a life-threatening
condition. Among domesticated animals, dogs are the most important species in the
epidemiology of this disease. In addition to becoming ill, dogs are reservoir hosts for L.
infantum, one of the two most important organisms in human visceral leishmaniasis .
Skin lesions and, rarely, visceral disease, have also been reported occasionally in other
domesticated animals, captive mammals in zoos, and wild animals.
Leishmaniasis is one of the most diverse and complex of all vector borne diseases.
Because it involves several overlapping species and sandfly vectors, the disease has a
complex ecology and epidemiology. It is caused by an obligate intramacrophage
protozoan, characterized by diversity and complexity. a total of about 21 Leishmania spp.
have been identified to be pathogenic to human . Leishmania are one of the several
genera within the family Trypanosomatidae , and are characterized by the possession of a
kinetoplast, a unique form of mitochondrial DNA. In most instances, they cause disease
in animals, and humans become infected incidentally when they enter an area of
endemicity. Leishmaniasis presents mainly in three clinical forms, of which visceral
leishmaniasis (VL) is the most severe form. Leishmaniasis has been considered tropical
afflictions that together constitute one of the six entities on the World Health
Organization/Tropical Disease Research (WHO/TDR) list of most important diseases.
The disease is endemic in 88 countries on five continents with a total of 350 million
people at risk and annually 12 million cases are reported. Of the 88 endemic countries, 22
are in the new World and 66 in the Old World with an estimated incidence of ~1.5 million
cases of cutaneous leishmaniasis (CL) and 500,000 cases of VL per year. More than 90%
of the CL cases occur in Iran, Afghanistan, Syria, Saudi Arabia, Brazil, and Peru. Of the
500,000 new cases of VL, more than 90% are reported from India, Nepal, Bangladesh,
southern Sudan and north-east Brazil. Despite this widespread geographic distribution,
human leishmaniasis is often very focal within an endemic area, leading to hotspots of
disease transmission. Leishmaniasis is transmitted through the bite of Phlebotomus
sandflies in the Old World and Lutzomyia in the New World . the disease is primarily
caused by Leishmania tropica in urban areas (dry type) and leishmania major (wet type)
in dry desert areas. CL is still considered and growing as an important health problem and
concern in especially the Mediterranean region, some countries of Africa, and almost all

countries of the Middle East, including Iran. The prevalence of infection is high in some
provinces of Iran such as Isfahan. Isfahan is a well-known endemic area of zoonotic
cutaneous leishmaniasis. In north east of Isfahan, the disease incidence is high.
Interventions for decreasing sand fly abundances and biting rates in domestic and peridomestic transmission foci, may reduce outbreak. In a study by Campbell-Lendrum et al.,
it was shown the significantly decrease in CL incidence in protected houses during the
trial. Also some activities such as working or helping in an agricultural area or water
collection could be associated with an increased risk of CL. Because most time of people
are spent in residential and work environment knowledge about health effects associated
with environment situation and human behavior in seems to be necessary. This study is
designed to determine association of domestic and extra domestic characteristics, human
behaviors and occupational activities with CL transmission.

Leishmanias
es
Kala zar

Neglected tropical diseases including leishmaniases

Sand fly shapes (female )

Morphology of leishmania parasite

1.AMASTIGOTES SHAPES

2.PROMASTIGOTES SHAPES

1. THE EATIOLOGY FACTOR :

Of all the insects that bite humans sand flies are probably the most widespread and
definitely one of the most irritating known over the world by a host of names
including sandflies, noseeums, no-see-ums, nicnics, nic nics, hop-a-long, biting midge,
punkie, punky, sandfly, sand flea, sand fly, black flies, black gnats, manta blanca,
palomilla, asa branca, quemadores and pringadores these are the colloquial names for the
small insects that bite and irritate and are capable of discomfort hugely disproportionate
to their size.

sand flies belong to the fly family Psychodidae, members of which are characterized by
their densely hairy wings which give them a moth-like appearance. Phlebotomines are
distinguished from other members of the family by the way they hold their wings above
the body in a vertical V.
There are about 700 species of phlebotomine sand flies of which about 70 are considered
to transmit diseases to people. The term sand flies is also sometimes confusingly used
for other small biting flies, especially ceratopogonidflies (biting midges) of the
genus Culicoides. It is used below exclusively for the Phlebotominae. Sand flies are
found mainly in the tropics with a few species also found in the temperate regions. They
occur in a wide range of habitats and species often have very specific habitat
requirements. In the Old World, leishmaniasis is found mainly in dry, semi-arid areas
whereas in the New World, this disease occurs mainly in tropical forests and savannas.
Sand flies (Phlebotominae) are blood suckers and their larvae inhabit places where there
is high organic matter such as in animal burrows, termite hills and tree holes.
Sand flies are best known as vectors of trypanosome species in the
genus Leishmania, causing diseases collectively known as leishmaniasis.
Ceratopogonidae
There are a thousands of variations of small biting insects but we are interested in the 1.5
- 4.0mm family of Ceratopogonidae who have piercing and sucking mouthparts although
only the female feeds on blood.
People in Australia are most likely to encounter sand flies in problem numbers around
tidal zones, lagoons, estuaries and mangrove swamps.
Sand fly bites often occur before the victim even realizes a potential risk. Small and hard
to see (no-see-ums) it may take hours or until the following day until an irritated, itching
local reaction occurs.
One old wives tale or myth suggests that sand flies create the annoying welts and lesions
seen on humans by urinating on them. Sand flies may well urinate on people but it does
not cause the common reaction seen on human skin. This is caused by the BITE.
Allergens:
The female sand fly bites humans in order to get protein from the blood - necessary for
egg laying and reproductive cycles. The bite involves the injection of saliva containing an
anti-coagulant, making it easier for the flea to draw blood from its host. The saliva
contains allergens that trigger the bodys immune system and red welts and lesions
develop. In Australia sand flies are unlikely to transmit disease although they are
problematic in more northern countries like the Philippines.
Sand fly activity is heaviest at sunrise and sunset (normal feeding times for most
things) and reputedly virulent nearing and on the full moon, although there is no evidence
to prove this. You are probably better off concentrating on other mythological phenomena
like werewolves and vampires around the full moon and just developing an overall
strategy for sand flies.
Affects Some More than Others:
Sand flies seem to affect some people more than others. Often one or two people will
react to the bites extremely badly while others in a group will only present with mild

irritation. It may be that humans are capable of developing a natural tolerance and
resistance with repeated exposure or it may be simply that one persons immune system
reacts differently than the next.
Many compounds are reported to be both repellent and cure for the bite of the sand fly
and many of the things on the list below have slipped into popular culture and bush
mythology.
sand fly Classification :
Class :Insecta
Order: Diptera
Family: Psychodidae
Order : dipetra
Genera :culicoides and phlebtomus
Species :phelobotomus papatasii
New World sand flies:

Genus: Brumptomyia
Genus: Lutzomyia. The only genus of phlebotomine flies that suck blood from
people in the New World.

Genus: Warileya

Old World sand flies:

Genus: Phlebotomus. The main genus of phlebotomine flies that suck blood from
people in the Old World and the only genus of phlebotomine flies that transmit
diseases to people in this region.
Genus: Sergentomyia. Feeds mainly on reptiles, sometimes transmitting the
protozoan parasiteSauroleishmania. Rarely bites people and does not transmit
diseases to people.

Life Cycle of sand flies :

It is difficult studying the life cycle of sand flies because the larvae are tiny and
don't live in well defined places, like mosquito larvae. The entire life cycle takes
20-40 days except in diapausing species (i.e. those that stop developing when
conditions become too cold).
Eggs. The female lays 30-70 eggs by scattering them around a potential breeding
site. They hatch within 1-2 weeks.
Larvae. Larvae feed on dead organic matter and are found in damp places
containing organic matter such as cracks in walls or rock, animal burrows and
shelters, caves, or in leaf litter. In regions with cool winters, larvae diapause in the
fourth (final) instar.
Pupae. Pupal development takes 5-10 days.
Adults. Emerge from the pupae in darkness, often just before dawn. Only the
female sucks blood, the food being used for egg production. Both males and

females feed on sugary secretions from plants or from honeydew produced by

homopteran bugs. Mating takes place at or near hosts: the males congregate in
leks on or near the host and produce sex pheromones. Females home in on hosts
using both host odor and the odor produced by the males. Vibration of the wings
by males can be important in encouraging females to mate.
Adults are mainly active in the early morning, evening and at night although they
can bite during the day if disturbed. When inactive, adult sand flies have habitatspecific resting sites that are characteristic of particular species. One of the main
ways in which entomologists study sand flies is by locating and studying them at
their resting sites. Resting sites are often similar or near to the larval breeding
sites and are usually places that are cool, humid and dark. Sand flies are able to
survive in dry environments by withdrawing to cool, humid resting sites during
the day and then becoming active at night when ambient temperatures drop and
humidity increases.
Seasonal activity of adults is affected mainly by temperature and rainfall.

Life cycle of sand flies

Disease organisms transmitted:

Leishmania. Protozoan parasites that cause visceral leishmaniasis (kala-azar) and

various types of cutaneous leishmaniasis (e.g. oriental sore, espundia) in people.
Bartonella bacilliformis. A bacterium causing the disease bartonellosis (Oroya
fever, Carrion's disease) in northwestern South America (Peru, Colombia and
Ecuador).

Sandfly Fever Virus. Transmitted by sand flies in North Africa and the Middle
East.

Toscana virus. Occurs in the northern and western Mediterranean.

Chagres and Punta Toro viruses. Occur in the New World.

Geographic Distribution:
Leishmaniasis has a long history. Designs on pre- Colombian pottery and the existence
of thousand-year old sculls with evidence of leishmaniasis prove that the disease has been
present in the Americas for a long time. It has also been present in Africa and India since
at least the mid-eighteenth century . Today, an estimated 12 million cases of leishmaniasis
exist worldwide with an estimated number of 1.5 -2 million new cases occurring
annually; 1 - 1.5 million cases of cutaneous leishmaniasis and 500000 cases of visceral
leishmaniasis .
The geographical distribution of leishmaniasis is restricted to tropical and temperate
regions, the living area of the sandfly. The leishmaniases are considered to be endemic in
88 countries (16 developed countries and 72 developing countries) on four continents.
Ninty percent of cases with cutaneous forms of leishmaniasis occur in Afghanistan,
Algeria, Brazil, Iran, Peru, Saudi Arabia and Syria, while ninety per cent of visceral
leishmaniasis cases are found in Bangladesh, Brazil, India, Nepal and Sudan
With the exception of Antarctica, Leishmania spp. have been reported on every continent.
These organisms are primarily endemic in tropical and sub-tropical regions and human
disease mainly occurs in Africa, parts of Asia, the Middle East, Latin America and the
Mediterranean region. In Europe, leishmaniasis appears to be spreading northward from
its traditional foci.
The distribution of each species of Leishmania affects the type of disease that occurs in
each region, as well as its severity. L. donovani causes visceral leishmaniasis in South
Asia and Africa. L infantum causes this disease in the Mediterranean, the Middle East,
Latin America and parts of Asia. Cutaneous leishmaniasis is caused by L. major in Africa,
the Middle East and parts of Asia, by L. tropica in the Middle East, the Mediterranean
and parts of Asia, and by L. aethiopica in parts of Africa. Many different species may be
involved in the Western Hemisphere, where cutaneous leishmaniasis can be found from
Mexico through South America.
In North America, limited foci of infection have been reported in Canada and the U.S.
Canine leishmaniasis caused by L. infantum and occurring mainly in Foxhounds has been
reported in a number of U.S. states and parts of Canada. Human cases have not been
linked to these animals. In addition, a focus of cutaneous leishmaniasis that has
sporadically affected humans or domesticated animals is found in south central Texas,
where a species of Leishmania (possibly a member of the L. mexicana complex) seems to
be endemic. Australia appeared to be free of Leishmania spp. until 2004, when this
organism began to be reported from captive kangaroos, wallabies and other marsupials.
Imported cases of leishmaniasis can also be seen in areas where Leishmania spp. are not
endemic. If appropriate insect vectors are not present, these organisms usually do not
become established in the country.

Distribution of leishmaniases over the world

Map 10.1 Global distribution of reported cases of leishmaniasis and

Leishmania/HIV co- infection

Transmission :
Leishmaniasis is a highly focal disease with widely scattered foci. The parasite may
survive for decades in asymptomatic infected people, who are of great importance for the
transmission since they can spread visceral leishmaniasis. Leishmania spp. are usually
transmitted indirectly between hosts by sandflies of the genera Phlebotomus and
Lutzomyia, which are biological vectors The parasites can also be transmitted directly
from person to person through the sharing of infected needles which is often the case
with the Leishmania/HIV co-infection. The disease has four main forms, depending on
the parasite species and the cellular immune system of the patient. Each species of
Leishmania is adapted to transmission in certain species of sandflies. Only the females
feed on blood. Sandfly activity occurs when it is humid, and there is no wind or rain.
These insects are usually most active at dawn, dusk and during the night, but they will
bite if they are disturbed in their hiding places (animal burrows, holes in trees, caves,
houses and other relatively cool, humid locations) during the day. They are attracted to
light and may enter buildings at night. Transovarial transmission of Leishmania does not
seem to occur, and in areas with cold temperatures, the parasite overwinters in
mammalian hosts. Other arthropods including ticks (Dermacentor variabilis and
Rhipicephalus sanguineus) and canine fleas may also act as mechanical vectors. Where
sandflies transmit Leishmania spp., ticks and fleas are probably unimportant in the
epidemiology of the disease; however, they might be involved in rare cases of dog-to-dog
transmission in other locations.
Mammals can be infected asymptomatically for long periods, and they often remain
chronically infected even after clinical cure. Subclinically infected animals can transmit
Leishmania to sandflies. These parasites have also been transmitted via blood
transfusions in people and dogs, and by transplacental transmission in dogs, mice and
humans. In canine leishmaniasis caused by L. infantum, the parasites can sometimes be
found in saliva, urine, semen and conjunctival secretions, as well as in blood. Venereal
transmission has been proven to occur in dogs, and other routes of spread might be
possible. Rare cases of horizontal transmission have been reported between dogs in the
same household or kennel. Case histories suggest that some of these animals might have
been infected during a fight. In other cases, a dog is known to have licked its
companions lesions or ingested blood during a hemorrhage. Epidemiological
investigations in U.S. Foxhounds also suggest that L. infantum has been transmitted
directly from dog to dog, although sandfly mediated transmission or other arthropodborne transfer has not been ruled out. In contrast, sandflies are thought to transmit the
disease to people from wild mammals in south-central Texas. The risk of direct
transmission from infected dogs to humans is unknown.
Epidemiology
Humans and domesticated animals are accidental hosts for many Leishmania spp., which
are maintained in cycles between wild animals and sandflies. L. infantum, L. peruviana
and possibly other species can be maintained in dogs, increasing the risk of transmission
to people. Other domesticated animals might be involved as secondary maintenance
hosts. L. donovani and L tropica are adapted to humans, but animals can also be infected
occasionally.
Disinfection

Leishmania spp. do not remain viable outside a host or in vitro culture. They can be
inactivated by 1% sodium hypochlorite, 2% glutaraldehyde, or formaldehyde. They are
also susceptible to heat of 5060C.
Infections in Humans
Incubation Period
People can carry some species of Leishmania asymptomatically for long periods, without
becoming ill. In humans, the reported incubation period for cutaneous leishmaniasis can
be as short as 1-2 weeks or as long as several months when it is caused by New World
species, and up to three years when Old World species are involved. The incubation
period for visceral leishmaniasis is 10 days to several years; most cases seem to become
apparent in two to six months.
Clinical Signs
Two forms of leishmaniasis, cutaneous and visceral, are seen in humans. Some texts also
distinguish a mucocutaneous form, while others consider it to be a subset of cutaneous
leishmaniasis. The form of the disease and the usual clinical signs vary with the species
of Leishmania. Some infections remain asymptomatic.
Cutaneous leishmaniasis
Cutaneous leishmaniasis often involves only the skin, and may be characterized by one to
dozens of lesions. Depending on the species of Leishmania, ulcers, smooth nodules, flat
plaques or hyperkeratotic wart-like lesions may be seen. The initial lesions, which occur
on skin that was exposed to sandflies, are usually papules. Many lesions remain localized,
but in some cases, the parasites may spread via the lymphatics and produce secondary
lesions on the skin, or occasionally the mucosa, of other parts of the body. Regional
lymphadenopathy sometimes occurs. Cutaneous leishmaniasis is usually painless unless
the lesions become secondarily infected, and except in the ear, the ulcers tend to remain
confined to the skin and do not affect the subcutaneous tissues. Most skin lesions heal
spontaneously; however, the speed of healing varies with the species of Leishmania. In
some cases, it may take several months to a year or longer. Some forms leave permanent
scars. HIV-infected individuals can have unusually severe cases, and the disease is more
difficult to cure. Steroid treatment or other forms of immunosuppression can also result in
unusually severe disease.
Disseminated leishmaniasis is a rare form of cutaneous disease. It is seen especially with
L. amazonensis in the Western Hemisphere, although other organisms can also be
involved. It also occurs in the Eastern Hemisphere, often in people who have concurrent
HIV infections. In diffuse cutaneous leishmaniasis, the nodules do not ulcerate but they
spread widely on the skin. They may cause damage to deep tissues, and can persist
indefinitely. The diffuse form can be incurable in some cases.
Leishmaniasis recidivans (lupoid leishmaniasis), another rare form, is characterized by
the development of new lesions around the edges of a healed skin lesion. It is most often
caused by L tropica or L braziliensis, and it does not heal without treatment.
Mucocutaneous
leishmaniasis (espundia) usually occurs in Latin America, where it
is caused by L. braziliensis braziliensis and, less often, by L. panamensis/ L. guyanensis.
Mucocutaneous leishmaniasis tends to occur 1 to 5 years after cutaneous leishmaniasis
caused by these organisms has healed, but it can also be seen while skin lesions are still
present. The initial signs are erythema and ulcerations at the nares, followed by
destructive inflammation that can spread to involve the nasal septum, and in some cases,
the pharynx or larynx. Frequent nosebleeds can be an early sign. The inflammation may
perforate the nasal septum, cause severe disfigurement of the face, or block the pharynx

or larynx. In some cases, the genitalia may also be involved. Mucocutaneous

leishmaniasis does not heal spontaneously.
Visceral leishmaniasis
Visceral leishmaniasis is usually an insidious, chronic disease among the inhabitants of
endemic areas; however, the onset may be acute in travelers from Leishmania-free areas.
In some cases (especially in Africa), a primary granuloma appears on the skin before the
systemic signs. The most common symptoms of visceral leishmaniasis are a prolonged
undulant fever, weight loss, decreased appetite, signs of anemia, and abdominal
distension with splenomegaly and hepatomegaly. Thrombocytopenia may cause bleeding
tendencies, including petechiae or hemorrhages on the mucous membranes, and
leukopenia can result in increased susceptibility to other infections. Other symptoms may
include coughing, chronic diarrhea, darkening of the skin, lymphadenopathy, and in many
cases, signs of chronic kidney disease. Mild cases with only a few symptoms may resolve
spontaneously. Unless they are treated, most other cases are eventually fatal, often from
secondary infections and other complications. Fulminant disease or atypical cases can
also occur, especially in patients co-infected with HIV. People with successfully treated
infections continue to carry the parasite, and the disease may recur if they become
immunosuppressed. Similarly, asymptomatically infected individuals may later develop
clinical signs.
Post-kala azar dermal leishmaniasis (PKDL) occurs after recovery in some cases of
visceral leishmaniasis caused by L. donovani. This syndrome is characterized by a
maculopapular, macular or-nodular rash around the mouth, which spreads. In Africa,
PKLD is common, usually occurs within 6 months of visceral leishmaniasis, and
typically disappears within a year without treatment. In South Asia, this syndrome is
relatively rare, occurs several years after visceral leishmaniasis has been cured, and
required prolonged treatment. In India, PKLD is seen in 1-3% of successfully treated
cases of visceral leishmaniasis.
Communicability
Leishmaniasis is usually vector-borne, but person-to-person transmission including
vertical (congenital) transmission, venereal transmission, and transmission by blood
transfusion has been reported. Newborns can be infected whether or not the mother was
symptomatic. Humans infected with some species of Leishmania can infect sandflies.
DIAGNOSIS OF LEISHMANIASIS :
Cutaneous leishmaniasis can be diagnosed by direct observation of the parasites in skin
scrapings, impression smears or skin biopsies stained with Giemsa, Leishmans, Wrights
or other stains. Amastigotes are easiest to find in recent or active lesions. Polymerase
chain reaction assays (PCR) are often used for diagnosis in areas where they are
available. Leishmania spp. can also be cultured. However, each species will grow only in
certain media, and some species can be difficult to isolate. Novy-MacNeil-Nicole (NMN)
medium, brainheart infusion (BHI) medium, Evans modified Tobies medium (EMTM),
Graces medium and Schneiders Drosophila medium might be used initially. Animal
inoculation into hamsters may also be valuable, especially with contaminated material.
Diagnosing leishmaniasis by in vitro culture requires 5 to 30 days, while animal
inoculation can take weeks or months. The species, subspecies and/or strain can be
identified by PCR, DNA hybridization, kinetoplast DNA restriction endonuclease
analysis, isoenzyme analysis, or immunological techniques that use monoclonal
antibodies. A delayed hypersensitivity test, the leishmanin skin test (Montenegro skin

test), is useful in the diagnosis of cutaneous and mucocutaneous leishmaniasis, but it is

usually negative in the diffuse cutaneous form. Antibodies are often slow to develop and
of low titer.
Visceral leishmaniasis can be diagnosed using some of the same techniques, including
direct observation of the parasites. Amastigotes may be found in peripheral blood, or
more often, in aspirates or biopsy smears from the spleen, bone marrow or lymph nodes.
PCR, culture or animal (hamster) inoculation may be particularly useful early, when
parasite numbers are low. Serology can also be helpful in this form of leishmaniasis.
Common serological tests used in humans include the immunofluorescent antibody test
(IFA), direct agglutination, enzyme-linked immunosorbent assay (ELISA), fast
agglutination-screening test (FAST), and a rapid immunochromatographic assay (K39
dipstick or strip-test). Other assays including gel diffusion, complement fixation, indirect
hemagglutination and countercurrent electrophoresis have also been used. Crossreactions can occur in some serological tests with leprosy, Chagas disease, malaria and
schistosomiasis. The leishmanin skin test/ Montenegro skin test is usually negative in
cases of visceral leishmaniasis, but reactions can be seen once the disease is cured.
Diagnostics of first episodes of VL have drastically improved in South Asia, but there are
still serious limitations for diagnostics that work in African contexts. The rK39 antigenbased rapid diagnostic test (RDT) can be used (alone) to confirm and exclude primary VL
in clinically-suspected patients in South Asia and is used in active case finding at
community level. But the test performs imperfectly in East Africa, with 80% to 90%
sensitivity.
Therefore, clinically suspect primary VL patients with a negative RDT in East Africa
need to be referred to a laboratory for further diagnosis by another serological test and/or
microscopic examination of spleen, bone marrow or lymph node aspirates. However, due
to physical barriers and insecurity, this referral is often not possible. Better, more
sensitive RDTs are needed for the African context
Examination of slides
such as :
1. biopsy specimens,
2. impression smears,
3. and dermal scrapings.
Provision of leishmanial culture medium
In vitro culture and PCR for diagnosis of leishmaniasis and species identification.
PCR does not require additional specimens besides the tissue obtained for culture .
Serologic testing using the rK39 Rapid Test, for detection of antibodies against
organisms in the Leishmania donovani species complex; useful primarily for
visceral leishmaniasis
CULTURE MEDIUM:
If possible, request culture medium before obtaining biopsy specimens or aspirates. CDC
can provide tubes of culture medium by overnight express mail. Keep the tubes
refrigerated until they are used; bring to room temperature shortly before inoculation.
Once inoculated, keep at room temperature and mail to CDC as soon as possible, by
overnight mail (preferably, for arrival within 24 hours of when the specimens were
obtained). Please do not ship on a Friday; we do not have weekend delivery service. Do
not ship cultures on cold packs or dry ice, but keep the cultures away from heat.

If culture medium was not obtained in advance, consult CDC to discuss the options
below:
If possible, place the specimen in a tube/vial that contains a sterile buffered medium
(e.g., buffered saline, RPMI, Eagles growth, Schneiders, Tobies), with a neutral pH
(~77.4). Use enough fluid to cover the specimen. Ship at room temperature, if the
specimen will arrive within 2448 hours. Otherwise, consult whether to refrigerate and
ship on a cold pack, to minimize the potential for overgrowth of skin flora
If no buffered medium is available, place the specimen in a sterile tube/vial; refrigerate
the specimen; and ship it on a cold pack, by overnight mail .
PREPARATION of the skin:
Inject anesthetic (e.g., 1% lidocaine with epinephrine 1:100,000) through intact skin
cleansed with 70% alcohol into the dermis underlying the area that will be sampled.
Avoid high concentrations of anesthetic, which could inhibit parasite growth in culture.
Thoroughly cleanse the pertinent area of skin (e.g., with 70% alcohol). It is preferable
not to use iodine because it could inhibit parasite growth in culture. If iodine is used, it
should be thoroughly washed off.
If biopsy specimens and/or dermal scrapings will be obtained, use a scalpel blade to
debride scabs and devitalized tissue from the relevant areas. Then apply pressure, with
sterile gauze, to achieve hemostasis.
BIOPSY specimens:
Obtain sterile, full-thickness, punch-biopsy specimens at the active border of the lesion.
Some practitioners recommend having the specimen include both affected and
unaffected tissue. Divide the specimen into 3 portions (or obtain multiple biopsy
specimens):
Use 1 sterile portion for leishmanial and other cultures (bacterial, mycobacterial, and
fungal); the portion placed in leishmanial culture medium also can be used for PCR.
Use 1 portion for impression smears .
Use 1 portion for histologic examination of tissue sections (fixed in 10% formalin;
embedded in paraffin) stained with H&E, Giemsa, and other special stainsto help
exclude mycobacterial, fungal, and other infectious etiologies.
Tissue IMPRESSION SMEARS :
Grasp the biopsy specimen with forceps. To avoid making a bloody smear, some
practitioners recommend briefly placing the cut surface on gauze or a paper towel to
remove excess blood. However, blotting the specimen might remove amastigotes that are
present on the surface.
Filet the specimen to increase surface area. Gently press the tissue with a rolling or
circular motion onto a glass microscope slide. Repeat in a parallel row down the slide.
Air dry the slide, fix it in methanol, and stain with Giemsa. Alternatively,
After making the smears, the tissue is not sterile but still is usable .
Needle ASPIRATES:
Draw up ~0.1 mL of preservative-free sterile 0.9% saline into a 1.03.0 mL syringe (the
better suction obtained with syringes at the larger end of the range may be advantageous).
For ulcerative skin lesions, insert the needle, through intact sterile skin, into the dermis of
the active border . Use a 23- to 27-gauge needle; small-gauge needles are particularly
useful for facial lesions.

Repeatedly move the needle back and forth under the skin, tangentially to the ulcer,
simultaneously rotating the syringe and applying gentle suction, until pink-tinged tissue
juice is noted in the hub of the needle. If necessary (if no aspirate is obtained), inject
0.050.1 mL saline under the skin and resume suction. After the aspirate is obtained,
withdraw the needle from the skin and discharge the aspirate into the leishmanial culture
medium (each aspirate into a different tube). Although thin smears of aspirates can be
made, they typically are suboptimal, unless a cytospin preparation is used.
Summary of TYPES OF SPECIMENS :
If possible, to increase sensitivity, use several techniques and obtain several specimens
per technique (e.g., from different lesions or different portions of the same lesion).
Preferentially sample areas that appear the youngest, most active, and least apt to be
superinfected.
Obtain sterile biopsy specimens for culture and PCR. Also use biopsy specimens for
impression smears (i.e., touch preparations) and histologic examination.
Obtain sterile lesion aspirates for leishmanial culture (PCR).
Of note, dermal scrapings can be obtained for Giemsa-stained thin smears. However,
obtain the dermal scrapings last, to minimize the risk of contaminating the site.
PREPARATION of the skin:
Inject anesthetic (e.g., 1% lidocaine with epinephrine 1:100,000) through intact
skincleansed with 70% alcohol into the dermis underlying the area that will be sampled.
Avoid high concentrations of anesthetic, which could inhibit parasite growth in culture.
Thoroughly cleanse the pertinent area of skin (e.g., with 70% alcohol). It is preferable
not to use iodine because it could inhibit parasite growth in culture. If iodine is used, it
should be thoroughly washed off.
If biopsy specimens and/or dermal scrapings will be obtained, use a scalpel blade to
debride scabs and devitalized tissue from the relevant areas. Then apply pressure, with
sterile gauze, to achieve hemostasis.
BIOPSY specimens:
Obtain sterile, full-thickness, punch-biopsy specimens at the active border of the lesion.
Some practitioners recommend having the specimen include both affected and
unaffected (e.g., nonulcerated) tissue. Divide the specimen into 3 portions (or obtain
multiple biopsy specimens):
Use 1 sterile portion for leishmanial and other cultures (bacterial, mycobacterial, and
fungal); the portion placed in leishmanial culture medium also can be used for PCR.
Use 1 portion for impression smears .
Use 1 portion for histologic examination of tissue sections (fixed in 10% formalin;
embedded in paraffin) stained with H&E, Giemsa, and other special stains to help
exclude mycobacterial, fungal, and other infectious etiologies.
Tissue IMPRESSION SMEARS (touch preparations):
Grasp the biopsy specimen with forceps. To avoid making a bloody smear, some
practitioners recommend briefly placing the cut surface on gauze or a paper towel to
remove excess blood. However, blotting the specimen might remove amastigotes that are
present on the surface.
Filet the specimen to increase surface area. Gently press the tissue with a rolling or
circular motion on to a glass microscope slide. Repeat in a parallel row down the slide.
Air dry the slide, fix it in methanol, and stain with Giemsa. Alternatively,

 After making the smears, the tissue is not sterile but still is usable (e.g., for PCR).
Needle ASPIRATES:
Draw up ~0.1 mL of preservative-free sterile 0.9% saline into a 1.03.0 mL syringe
(the better suction obtained with syringes at the larger end of the range may be
advantageous).
For ulcerative skin lesions, insert the needle, through intact sterile skin, into the dermis of
the active border . Use a 23- to 27-gauge needle; small-gauge needles are particularly
useful for facial lesions.
Repeatedly move the needle back and forth under the skin, tangentially to the ulcer,
simultaneously rotating the syringe and applying gentle suction, until pink-tinged tissue
juice is noted in the hub of the needle. If necessary (if no aspirate is obtained), inject
0.050.1 mL saline under the skin and resume suction. After the aspirate is obtained,
withdraw the needle from the skin and discharge the aspirate into the leishmanial culture
medium (each aspirate into a different tube).
Although thin smears of aspirates can be made, they typically are suboptimal, unless a
cytospin preparation is used.
Dermal SCRAPINGS: (for thin smears):
As discussed above: first thoroughly debride the relevant portions of ulcerative lesions;
then apply pressure to ensure good hemostasis (to avoid making a bloody smear). A
convenient location for obtaining specimens from ulcerative lesions is the area
immediately adjacent to or beneath the active border (e.g., beneath the necrotic lip of the
lesion).
Slit-skin smear technique: Some practitioners first make an incision before obtaining a
scraping. For this technique, pinch the skin to exclude blood and use a scalpel blade to
incise a several millimeter long and deep slit through intact skin into the upper dermis.
For ulcerative lesions, consider starting the incision in the active border and proceeding
radially out from the ulcer, across several millimeters of intact skin. (This technique also
can be used for nodular lesions.)
Obtain tissue juice and flecks of tissue by scraping the upper dermis (e.g., beneath the
necrotic lip of the lesion or along the walls of the incision, if one was made in the skin)
with a sharp instrument (e.g., a scalpel blade or stainless steel spatula). After obtaining as
much tissue pulp as possible, make as thin a smear as possible. Air dry the slide, fix it in
methanol, and stain with Giemsa. Alternatively, can fix/stain the slide. Although dermal
scrapings also can be cultured, the risk for contamination is high

Treatment
Visceral or cutaneous leishmaniasis can usually be cured in immunocompetent
individuals. Pentavalent antimonials can be used where the parasites are sensitive to these
drugs, but resistance is a major problem in some areas. Other drugs such as allupurinol,
amphotericin B or liposomal amphotericin B, and miltefosine may also be used. Most of
the drugs used to treat leishmaniasis must be given parenterally. Visceral leishmaniasis in
AIDS patients is often resistant to treatment, and many patients relapse.
Cutaneous leishmaniasis may be treated to speed healing, decrease scarring and decrease
the risk of mucosal disease or relapse. Intralesional, topical or systemic drugs may be
used, depending on the species of Leishmania and the risk of more serious complications.
Cryotherapy, thermotherapy, or curettage have also been employed in some cases. Some
cutaneous leishmaniasis lesions that are improving may simply be observed, if they are
caused by relatively benign organisms. Mucosal leishmaniasis is a serious condition and
it is treated with systemic drugs.
Prevention
Preventative measures against sandflies include using insect repellents such as DEET,
covering exposed skin, and staying on higher floors of buildings in the evening or at
night, as these insects are poor fliers. Fans can also be helpful, and insecticidal sprays can
be used to kill the insects inside houses. Insecticide-treated bed nets decrease bites from
these insects at night. Untreated bed nets are not generally useful: sandflies are very tiny
and can pass through the mesh of most nets, while bed nets with a very narrow mesh may
be too hot in warmer climates. Insecticide-treated bed sheets, window curtains and slowrelease paint have also been used. Insecticide spraying programs have been conducted in
some countries.
Treatment of human patients may be helpful in areas where anthroponotic transmission is
important. Decreasing the incidence of L. infantum in dogs can help protect people from
this organism. Some studies have shown that insecticide-impregnated dog collars
protected both dogs and children in areas where they were used. Infected dogs have been
culled in some countries; however, there are doubts about the efficacy of these programs,
and in some countries, such programs would also not be accepted. Many species of
Leishmania, particularly species that cause cutaneous leishmaniasis, have wild animals as
their reservoir hosts. The only practical way to decrease the incidence of these diseases is
personal protection with insect repellents and other measures.