Review
Leishmaniasis: a re-emerging zoonosis
Rosanna Marsella, DVM, DipACVD, and Rafael Ruiz de Gopegui, DVM, PhD
From the Department of Small Animal Clinical Sciences, Virginia
Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia,
and Department of Pathology and Animal Production, Veterinary Faculty,
Autonomous University of Barcelona, 08193 Bellaterra (Barcelona),
Spain
CorrespondenceRosanna Marsella, DVM, DipACVD, Department of Small Animal Clinical
Sciences, PO Box 100126, College of Veterinary Medicine, University of
Florida, Gainesville, FL 32610
Introduction
Leishmaniases are a group of infections caused by protozoa
of the genus Leishmania. With some exceptions, leishmani
ases are zoonotic diseases with transmission between sand
fly vectors and mammalian reservoirs in sylva tic or peri
domestic cycles. I Leishmaniases occur in more than 80
countries, and it is estimated that more than 15 million
people are infected, with more than 400,000 new human
cases every year. 2 Estimates are not accurate, however, as
leishmaniasis is a reportable disease in only 30 countries,
and a large number of infections are asymptomatic or
"subclinical." Human visceral leishmaniasis is still endemic
in the Mediterranean basin, and epidemics of the classical
visceral leishmaniasis are ongoing in India, Brazil, Sudan,
South America, North Africa, and central Africa. New
clinical presentations have been reported in American
military veterans acquired during Operation Desert Storm)
The emergence of leishmaniasis as an opportunistic infec
tion in people infected with human immunodeficiency virus
(HIV) has been reported in southern Europe, and it is
possible that leishmaniaI parasites of lower mammals could
also produce disease in humans afflicted with acquiredimmunodeficiency syndrome (AIDS).4
In the USA, imported leishmaniasis is occasionally dia
gnosed. Recently, an enzootic transmission cycle involvingLeishmania mexicana in Texas has been described, indicat
ing that cutaneous leishmaniasis may be more commonthan previously thoughr.! The purpose of this article is
to review, in a comparative manner, the host-parasite
interactions, pathogenesis, clinical signs, and therapy of
leishmaniasis, and to discuss the role of animal reservoirsin human disease.
© 1998 Blackwell Science Ltd
Classification of the agent
Leishmania spp. are intracellular protozoans belongingto the family Trypanosomatidae, order Kinetoplastida.Members of this genus infect primarily mammals, although10 species have been found in lizards in the Old World. Inthe last two decades, the classification of Leishmaniaspp. has been changed following the development of newtechniques.f One of the most useful classification methodsis based on the location of enzymatically active proteins(isoenzymes) via electrophoresis. Zymodemes are definedas groups of Leishmania organisms that share the sameelectrophoretic isoenzyme migration patterns.! The isoenzyme pattern obtained from patient isolates is comparedwith reference strains.
Morphology of l.eishmania parasites
Leishmania is heteroxenus and completes its life cycle usingtwo hosts. It is characterized by three different morphologictypes: amastigote, promastigote, and paramastigote. Theamastigote is found in the cells of the vertebrate hosts, andthe promastigote and paramastigote in the intestine of thesand fly and in culture.
The amastigote stage (2.5-5.0 x I.5-2.0 um) is usuallyfound within' macrophages and other cells of the reticuloendothelial system of the vertebrate host. A special mitochondrial structure containing deoxyribonucleic acid(DNA), called a kinetoplast, is present adjacent to thenucleus. A flagellum is not present (Figs 4-7).
Promastigotes are the form most commonly found in theinvertebrate vector. They are characterized by a spindleshape (14-20 X I.5-3.5 um) and a flagellum in theanterior part. 801
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802 Review Leishmaniasis
Figure 1 Attachment between amastigotes and macrophageand interaction between T cells and parasite
Figure 2 Granulomatous lesion in the nasal region of a 7year-old crossbred female dog. The lesion appeared somedays after a cat scratch
Figure 3 Same case as in Fig. 2, I month after therapy usingN-methyl glucosamine 100 mg/kg/day s.c. and allopurinol20 mg/kg p.o. b.i.d.
International Journal of Dermatology 1998,37, 801-814
Marsella and Ruiz de Gopegui
Paramastigotes are also found in the invertebrate vector.This is a transitional stage between the previous twomorphologic types.
The vector
Numerous phlebotomine sand flies have been identified asvectors for the transmission of Leishmania (Table I). Theincrimination of a particular sand fly as vector is difficultas the presence of Leishmania stages in the gut doesnot necessarily mean that the insect is able to transmitthe disease."
The taxonomy is described below.Phyllum: ArthropodaClass: InsectaOrder: DipteraSuborder: NematoceraFamily: PsychodidaeSubfamily: PhlebotominaeFive genera comprising over 600 species that are distributedthroughout the tropics, subtropics, including rain forests,deserts, and highlands:Sergentomyia spp.Phlebotomus spp.Lutzomya spp.Brumptomyia spp.Warileya spp.
Sand flies are nocturnal insects that feed on mammals,birds, and reptiles according to the particular species. Onlythe female is hematophagous (blood-sucking) and a bloodmeal is required to lay eggs. The bite is usually painful andcauses a papular reaction that may persist for weeks. Sandflies are more common during warm months. Their flightrange is very limited (estimated to be 250 m). Sand fliespass though ordinary mesh screens due to their small size(2-5 mm). Under favorable conditions, the life cycle canbe completed in as little as 41-58 days. The life stagesinclude eggs, larvae, pupae, and adults. Females lay around100 eggs on the ground that hatch in 1-2 weeks. Larvaerequire a constant temperature, complete darkness, andorganic material to mature.
Life cycle of Leishmania spp.
The cycle starts when a sand fly takes a blood meal on aninfected vertebrate, ingesting amastigotes present in thedermis. Transformation from amastigote to promastigoteoccurs within 24-28 h. Parasites may either be free in theintestinal lumen of the sand fly or attached to the wallsby hemidesmosomes. Localization of the parasite in theintestine varies according to the particular species of vectorand the strain of Leishmania. This feature has been usedin the past as a criterion of classification of Leishmania spp.?
© 1998 Blackwell Science Ltd
Marsella and Ruiz de Gopegui
Figure 4 Leishmania infantumamastigotes in a fine needleaspirate from nodule shown inFig. 2. Dot-ELISA was negative.Diff-Quik stain, X 1000
magnification
Figure 5 Leishmania infantumamastigotes in a fine needleaspirate of lymph node. Diff-Quikstain, X 1000 magnification
After replication in the intestine, promastigotes migrate tothe esophagus and pharynx.
Following a sand fly bite, between 10 and 200 promastigotes present within the proboscis are regurgitated andpenetrate the dermis. Sand fly saliva plays a role in establishing the infection by reducing nitric oxide (NO) production by activated macrophages.f In heavily infected sandflies, the proboscis is congested with parasites, makingfeeding difficult and leading to repeated bites and multipleinoculations." Promastigotes do not actively migratetowards macrophages; they remain in the intercellularspace and activate complement by an alternative pathwaytriggering the accumulation of neutrophils and macro-
© 1998 Blackwell Science Ltd
Leishmaniasis Review 803
phages. Many promastigotes are thought to be destroyedby polymorphonuclear leukocytes and a few transforminto arnastigotes.? Adhesion between the parasite andmacrophages is a fundamental step for invasion of the hostcell. Numerous receptors have been identified on the surfaceof Leishmania, among which glycoprotein 63 (gp63) andlipophosphoglycan (LPG) are the most important. Bothserum-dependent and serum-independent mechanisms ofadhesion occur, and complement receptors (complementreceptors I and 3) are used by the parasite to bind to
macrophages."? Once adhesion has occurred, Leishmaniaparasites are engulfed and enter the phagolysosome. Leishmania spp. have developed several mechanisms to resist
International Journal of Dermatology 1998,37, 801-814
804 Review Leishmaniasis
the digestive and antimicrobial activity of the phagocyticcell (Table 2). Amastigotes are more resistant than promastigotes to cytokine-induced, oxygen-dependent antimicrobialmechanisms, reflecting an adaptation to intracellulargrowth. II Amastigotes multiply by binary fission insidemacrophages, leading to their rupture. 10
Epidemiology of the disease and reservoirhosts
With the notable exception of L. tropica and L. donovani,leishmaniases are zoonotic diseases. In most cases, morethan one mammalian species is found to be infected
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Marsella and Ruiz de Gopegui
Figure 6 Leishmania infantumamastigotes in the spleen of a5-year-old male intact MiniatureSchnautzer. The dog had severerenal failure and was subjected toeuthanasia due to the poorprognosis. Diff-Quik stain, X IOOOmagnification
Figure 7 Leishmania infantumamastigotes in the bone marrowfine needle aspirate of a I -year-oldmale intact Boxer. Dog wasclinically normal, but urinalysisrevealed proteinuria and a protein!creatinine ratio over normal limits.Diff-Quik stain, X IOOOmagnification
with a given species of Leishmania (Table I), making theidentification of a specific reservoir host difficult. In theMediterranean basin, infection is mainly caused by Leishmania infantum. Dogs have long been recognized as thereservoir host. The prevalence of infection is much higherin dogs (20-40%) than in humans (I-2%), and the diseaseis only sporadically reported in humans, even in areasendemic for canine leishmaniasis. The evaluation of theintradermal reactivity to the leishmanial allergen (leishmanin) in humans living in endemic areas showed that theprevalence of positive reactions increased with age, reaching43% in individuals between 45 and 55 years of age,suggesting that humans are frequently exposed to parasites,
© 1998 Blackwell Science Ltd
Marsella and Ruiz de Gopegui
Figure 8 A y-year-old male intact Great Dane with advancedleishmaniasis and concurrent babesiosis. Terminal cachexiaand marked abdominal distension due to hepatomegaly andabdominal effusion are evident
but that they develop resistance. 12,13 While the role ofthe canine population as a reservoir for human visceralleishmaniasis (L. infantum) in the Mediterranean basin hasbeen established, a reservoir has not been identified forstrains of L. infantum that cause cutaneous leishmaniasisin humans. These strains have so far been isolated onlyfrom humans.
In the New World, numerous wild and domestic animals,including dogs, cats, horses, and donkeys, may serve asreservoir hosts, allowing the transmission of infection insylvatic and domestic cycles. Humans are considered to beaccidental hosts developing both cutaneous and mucosallesions (Table I). Leishmaniasis has also been reported inthe USA. Cases have been described in humans, dogs(Oklahoma, Ohio, Florida, and Texas), and in one cat,leading to the conclusion that the disease is more commonthan originally thought. I 4- 16 Cutaneous leishmaniasis(L. mexicana) is endemic in Texas, and 20 new cases inhumans were described in this state between 1980 and1989.5 The distribution of cases followed the distributionof wood rats (Neotoma micropus) , suggesting that theseanimals are the reservoir hosts. The annual prevalence ofLeishmania mexicana in wood rats using the polymerasechain reaction varies from 5.6 to 27%.5 Lutzomya anthophora, a sand fly associated with wood rat burrows, wasinitially thought to be the vector. Alternatively, Lutzomyadiabolica, an avid human feeder that has also been foundin association with wood rats, may serve as a vector.
Immunology
Susceptibility to the disease depends on the T-cell andmacrophage response. The murine model of infection withLeishmania major suggests that, of all the T-cell subpopula-
© 1998 Blackwell Science Ltd
Leishmaniasis Review 805
tions, T-helper (Th) cells play a fundamental role in theoutcome of leishmaniasis. Expansion of the Th1 subsetwith the production of interleukin 2 (IL-2) and gammainterferon (y-IFN) leads to the resolution of infection inresistant mice, while Th2 proliferation and IL-4 productionleads to the progression of the disease in susceptibleanimals. Great effort has been made to investigate theregulatory cells and molecules involved in the differentiation of T cells from Tho precursors to the Th1 and Th2subsets, but the exact mechanism is still largely unknown.Factors that may play a role include antigen-presentingcells, antigenic load, type and amount of cytokines presentat the time of inoculation of parasites, and the specificleishmanial antigen.t-"?
In dogs, susceptibility to the disease is due to a lack ofa specific Tcell response (assayed by both intradermal
skin test and proliferation assays) and to a notable, butineffective, humoral response, as previously demonstratedfor mice, guinea pigs, and humans. I 8, I 9 Suggestive evidence
of suppressed cell-mediated immunity also comes from the
observation of high numbers of Demodex mites in dogswith leishrnaniasis.t? Demodex mites are present in low
numbers in the skin of normal individuals, but if immunosuppression occurs they increase in number.
Leishmaniasis and immunosuppression
Since the mid-r oSos, there has been a dramatic increase inthe number of cases of leishmaniasis in HIV-infectedpatients. A synergistic relationship between leishmaniasisand AIDS exists. Specifically, it is now known that promastigotes trigger tumor necrosis factor-alpha (TNF-a) synthesis
(89% of patients with visceral leishmaniasis have elevatedlevels of circulating TNF-a),21 and this cytokine has theability to upregulate HIV virus replication.'? This notion
is supported by data indicating that 3-7% of HIV-positiveindividuals in southern Europe develop visceral leishmaniasis. In these areas, more than 50% of visceral leishmaniasisin adults is now related to HIV infection."
HIV-positive individuals are not only more susceptibleto leishmaniasis, but also develop visceral disease evenafter infection with dermotropic strains of Leishmania,suggesting that tropism of the parasite is largely influencedby the immune status of the host. Progression from seropositivity to AIDS and visceralization of leishmaniasis involvea relative switch from a Th1 to a Th2 response, andthis explains why even seropositive asymptomatic HIVindividuals may develop a visceral dissemination of dermotropic strains. Recent data show that visceralization occurswith the same frequency in both asymptomatic seropositiveindividuals and in AIDS cases.P
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806 Review Leishmaniasis
Clinical signs: a comparative approach
The concept that each species of Leishmania causes adistinct clinical syndrome is no longer valid. Infection withLeishmania can either lead to clinically silent disease or toa spectrum of clinical signs depending on the immunologicstatus, genetic background and nutritional status of thehost, the number of parasites and their virulence factors,the site of inoculation, the composition of sand fly saliva,and other variables that have yet to be elucidated. I Sandfly saliva containing vasodilators favors the infection ofcirculating monocytes and visceralization, whereas salivathat triggers minimal vasodilation will lead to dermalinfection only.'
Leishmaniasis in humans has been traditionally classifiedas cutaneous, visceral, and mucocutaneous (Table 1).22
I Cutaneous form. New World: L. braziliensis, L. mexicana; Old World: L. major, L. tropica, L. aethiopica,L. infantum.
2 Visceral form. L. donovani, L. infantum.3 Mucocutaneous form. L. braziliensis, L. aethiopica.In dogs, manifestations of the disease are quite variable.v'After infection, various outcomes are possible (Table 3).I Clinically silent infection.2 Cutaneous manifestations.3 Non-specific signs.4 Multisystemic disease.
Clinical pathologic findings
Hematologic and serum biochemical data vary with theclinical presentation of the disease (Table 4).24
International Journal of Dermatology 1998,37,801-814
Marsella and Rui: de Gopegw
Figure 9 Ehrlichi aplatys morula ina platelet. Buffy coat smear stainedwith Diff-Quik, x 5000
magnification
Pathologic findings
Leishmania spp. amastigotes have been described withinmacrophages in spleen, lymph nodes, tonsils, bone marrow,liver, urinary bladder, intestinal lamina propria, lung,choroid plexus, cerebrospinal fluid, urine, and semen, andalso in phagocytic cell types other than macrophages, suchas monocytes, neutrophils, eosinophils, endothelial cells,and fibroblasts.s! Dogs with an anergic immune systemdevelop generalized distribution of the parasite and achronic and profuse cellular infiltration with macrophages,lymphocytes, and plasma cells. The cells form either adiffuse infiltrate or poorly organized microgranulomata.Animals that are either resistant to the infection or asymptomatic carriers tend to develop highly organized epithelioidgranulomata, evidence of an effective cell-mediatedimmunity. 26
Diagnosis (Table 5)
Parasitologic diagnosis
Amastigotes can be demonstrated in Giemsa-stained smearsfrom tissue aspirates (Figs 4-7). The kinetoplast is usefulin differentiating Leishmania from intracellular pathogenswith similar morphology. Definitive diagnosis has beentraditionally based on cytologic or histopathologic observation of amastigotes. Immunoperoxidase staining is moresensitive for the identification of amastigotes in infectedtissues."? Recently, the application of polymerase chainreaction (PCR) technology has allowed the identificationof Leishmania spp. to a higher degree of sensitivity. Polymerase chain reaction using primers from the LeishmaniarRNA gene allows the identification of parasites from a
© 1998 Blackwell Science Ltd
Marsel/a and Ruiz de Gopegui Leishmaniasis Review 807
Table 1 Synopsis of the most common types of Leishmania, their geographic distribution, reservoir hosts and type of diseasecaused in humans
Agent Vector Geography Animal reservoir Humans
L. braziliensis complex Lutzomya longipalpis Argentina Forest rodents (Proechimys Mucocutaneous form.
L. braziliensis braziliensis Phlebotomus yucumensis, Bolivia guyanensis) and marsupialia Skin lesions including papules,
L. braziliensis guyanensis P. I/anosmartinsi (Oidelphi marsupialis) have been nodules, and ulcers. Mucosal
L. braziliensis panamensis Lu. umbratilis, Brazil identified as reservoirs, while lesions in the mouth, nose,
Lu. flaviscutel/ata humans are accidental hosts. In pharynx, and larynx leading to
Lu. spinicrassa Colombia Brazil, the main reservoir host in destruction of cartilage and
Lu. trapidoi Boyaca, Caldas the sylvatic cycle has not been disfigurement (espundia) were
Lu. migonei, Lu. gomezi Venezuela identified, but domestic dogs very common in the past (>80%).
(Canis familiaris), horses (Equus At the present time, this form
cabal/us), donkeys, and rats may occurs in less than 5% of cases,
serve as reservoir hosts in the and cutaneous lesions are more
urban cycle. Sloths (Chloepus common. Visceral involvement is
didactylus) , anteaters, and not usually seen, but death may
opossums are the main reservoirs occur due to secondary bacterial
for L. guyanensis. Sloths infections
(Chloepus hoffmannii and
Bradypus infuscatus) are the
major reservoir for L. panamensis
L. mexicana complex Lutzomya anthopora, USA (Texas), Various rodents have been Mucocutaneous form.
L. mexicana amazonensis Lu. diabolica, Bolivia, Brazil, identified as reservoir. Humans Small erythematous papules
L. mexicana pifanoi Riphicephalus sanguineus? Colombia, are accidental hosts. Wood rat occur at the site of the insect bite
L. mexicana aristedesi (Texas), Lutzomya Ecuador, (Neotoma micropus) is the and slowly develop into an
L. mexicana garnhami olmeca olmeca (Belize) Guatemala, reservoir of infection in Texas ulcerated nodule. Eventually, the
L. mexicana venezuelensis Mexico, Peru, lesions heal, leaving a depressed
Venezuela, Belize scar (ears are involved in 40-55%
of cases as they are common
areas of Lutzomya bites). In
Texas, cutaneous nodules are the
most common clinical
presentation. In Belize, this
infection is limited to people
entering the forests
L. enrietli Lutzomya gasparviannai Bahia, Espirito Domestic guinea pigs (Cavia , Human infection not reported
Santo porcel/us) develop ulcers in the
skin and may act as a reservoir.
No visceralization occurs
L. hertigi P. claustrei, P. davisi, Porcupines may act as reservoir. No human infection ever reported
P. chagasi, P. maripaensis No canine infection has ever been
reported
L. chagasi (Cunha and Lutzomya longipalpis Bolivia, Brazil, Foxes are the reservoir hosts in Visceral form.Chagas) Colombia, EI the sylvatic cycle, while dogs and Incubation may last for weeks and
Salvador, opossums (Oidelphi marsupialis) months. Subacute fever,
Honduras, are the hosts in the peridomestic splenomegaly, pancytopenia, and
Mexico, cycle wasting are common clinical
Venezuela presentations
L. peruviana Lutzomya noguchii, Peru, Bolivia Domestic dog (Canis familiaris) is Two different clinical forms are
Lu. verrucarum, the natural host recognized: the "cutaneous
Lu. peruensis Andean form" called uta and the
"Amazonian jungle form" with
mucous membrane involvement
resembling espundia
© 1998 Blackwell Science Ltd International Journal of Dermatology 1998,37,801-814
808 Review Leishmaniasis
Table 1 Cant.
Marsella and Ruiz de Gopegui
Agent
L. donovani
L. infantum
L. tropica
Vector
P. ariasi
p. longicuspis
P. major
P. orientalis
P. perniciosus
P. smtmovi, P. longiductus
P. tobbi
P. martini
P. argentipes
P. chinensis
P. pemiciosus, P. longicuspis.
P. chinensis, P. mongolensis,
P. caucasicus
P. perfiliewi. P. pappatasi,
P. serqent), P. longicuspis
Geography
France
Yemen. Ethiopia,
SomaliaSoutheast Asia
SudanItaly, North Africa
Russia
Cyprus
Kenya
India. Afghanistan
China
Albania. Algeria.
China. Egypt.
France, Greece,
Israel, Italy,
Pakistan,
Portugal, Spain,
Turkey,
Oklahoma (USA)
Afghanistan,
Greece. Kuwait,
Turkey, Yemen
Animal reservoir
Visceral leishmaniasis caused by
L. donovani in India is not thought
to have an animal reservoir, and
parasites can be isolated from
normal-appearing skin of patients
up to 4 years after treatment
Dogs are more commonly
affected (20-40%) than humans
(1-2%) in endemic areas. Other
hosts are jackal, fox, wolf,
porcupine, fennec fox. and rat.
The common rat can also be a
reservoir. In dogs, clinical signs
may range from inapparent to
multisystemic disease. Diagnosed
in a few cases in the USA in dogs
imported from Greece. Spain, and
Portugal
It is thought not to have an animal
reservoir and to be an
anthroponosis. Rats may act as a
reservoir. Isolation of parasites in
the skin of some dogs is raising
the possibility that dogs may be a
reservoir in some areas.
Humans
Visceral form in humans (kala
azar, black fever, dum-dum fever).
After several months of
incubation. irregular fever and
liver and spleen hypertrophy arenoted. Mortality is 75-95% if
untreated. Post-kala-azar dermal
leishmaniasis can occur (up to
20% of cases) after treatment of
the visceral form. and may serve
as a reservoir for infection.
Macules, papules, nodules. and
plaques are numerous
Viscerocutaneous form.It causes Mediterranean or
infantile kala-azar (90% of
affected people are children less
than 5 years old). Cutaneous
lesions are usually more
inflammatory and more likely to
be solitary than those caused by
L. major
Cutaneous form.
Oriental sore (dry). Aleppo button,
Jericho boil, Pendinsk ulcer.
Ulcers and sores in exposed parts
of the body leading to permanent
scars. Rarely fatal. Leishmania
recidivans has been associated
with L. tropica. Typical lesions are
red plaques with spreading
borders and healing centers
L. major P. duboscqi.
P. pappatasi.
P. salehi,
P. caucasicus
Senegal. Egypt, Natural hosts are the desert
Southeast Asia. rodents such as the great gerbil
India, Afghanistan. (Rhombomys opimus). gerbil.
Israel, Kuwait, suslik, sand rat. hedgehog, and
Pakistan. Yemen mustelid carnivores. Infection in
dogs has never been reported
Cutaneous form (wet form of the
oriental sore). It starts as a small
papule, which appears
immediately after the insect bite
or after 2-4 weeks. Papule
enlarges in size. and becomescrusted in the center. When crust
is removed. an ulcer with
ulcerated borders is found.
Subcutaneous nodules develop
along the iymphatics. After 6-12
months, the lesion heals leaving a
depressed scar. No involvement
of mucous membranes. Lymph
nodes are often involved. In some
cases, lesions are active for more
than 24 months (non-healing
chronic cutaneous leishmaniasis)
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Marsella and Ruiz de Gopegui
Table 1 Cont.
Leishmaniasis Review 809
Agent
L. aethiopica
Vector Geography
Ethiopia, Kenya.
Sudan
Animal reservoir Humans
Cutaneous form.Similar to L. major. In some
cases. it causes widespread skin
lesions that resemble lepromatousleprosy
Table 2 Adhesion and escape mechanisms of Leishmania spp.
Leishmania
Lipophosphoglycan
Glycoprotein 63
Superoxide dismutase
Acid phosphatase
Mechanism of interaction with the host
Binds lectin-like binding sites on CR3 and mediates adhesion to macrophages
Inhibits insertion of C5b-9 complex in the membrane preventing lysis
Impairs generation of respiratory burst through inhibition of protein kinase C
Inhibition of lysosomal ~ galactosidase
Oxygen radical scavenger
Protease capable of degrading lysosomal enzymes
Degradation of H20 2
Inhibition of oxidative burst
variety of samples, including human and canine bonemarrow, lymph nodes, skin biopsies, and heparinizedwhole blood.r"
Immunologic diagnosis
This involves the detection of antibodies against Leishmaniaparasites or a specific cell-mediated immune response. Acell-mediated response can be detected by measuring thecytokine production or proliferative response of peripherallymphocytes to Leishmania antigens or delayed hypersensitivity to intradermal injection of leishmanial antigens. Currently, a Leishmania antigen is not approved by the Foodand Drug Administration for intradermal skin testing inthe USA. The identification of anti-leishmania antibodiesin serum has been attempted by several methods; however,cross-reaction between different species of the genus Leishmania with Trypanosoma cruzi and Babesia spp. is possible.Serologic testing should be attempted if visceral leishmaniasis is suspected and the parasitologic diagnosis is negative.A positive serologic titer, however, does not necessarilyimply that clinical leishmaniasis will develop. Serology maybe negative in patients with visceral leishmaniasis secondaryto AIDS. In those cases, the detection of parasites by culture,microscopy or polymerase chain reaction is necessary toachieve a diagnosis of visceral leishmaniasis. 29 In localizedcutaneous leishmaniasis, serologic diagnosis is unreliablein both dogs and people and direct diagnosis is required.t?
© 1998 Blackwell Science Ltd
The following tests have been developed.I Indirect immunofluorescent antibody test (IFAT) shows
a high specificity and sensitivityY2 Enzyme-linked immunosorbent assay (ELISA) is rou
tinely used in people with visceral disease'> and in dogs.A modification of this method, dot-ELISA, constitutes asimpler immunoassay.t"
3 Immunoblot analysis (Western blot) has a high sensitivityfor the detection of antibodies in human visceral leishmaniasis and canine Ieishmaniasis.H
4 Direct agglutination test (DAT) has a similar specificity toIFAT, but a lower sensitivity in both dogs and humans.U
5 Indirect immunoperoxidase assay (IPA) is more sensitiveand specific than IFAT for the diagnosis of visceralleishmaniasis in people.
6 Recombinant Leishmania infantum proteins (rLIP2a andrLIP2b) have been studied as potential tools for thediagnosis of human mucocutaneous and visceral leishmaniasis. Leishmania P2 acidic ribosomal proteins arerecognized by the sera of some patients with leishmaniasis, avoiding cross-reactivity with sera from patientswith systemic lupus erythematosus and Chagas' disease.t"
Therapy
There is no universally effective therapy for leishmaniasis.Canine leishmaniasis usually has a poorer prognosis than
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810 Review Leishmaniasis
Table 3 Clinical signs commonly reported in dogs and in humans with leishmaniasis
Humans
Marsella and Ruiz de Gopegui
Dogs
Cutaneous
Systemic
Localized
Disseminated
Mucosal
L. recidivans
Post-kala-azar dermal
leishmaniasis
Mononuclear phagocyte
system
Gastrointestinal
Respiratory
Ocular
Others
Concomitant infections
Single papule evolving into a noduleand then into an ulcer,
Non-ulcerative soft nodules,
resembling leprosy
Occurs only in a small percentage
after the occurrence of
localized cutaneous disease
(L. braziliensis). Nodules in
nasal or pharyngeal mucosa
Caused by L. tropica. Slowly healing
ulcer
Caused by L. donovani (India),
usually after treatment of the
visceral disease. Macules, nodules,
and plaques
Hepatosplenomegaly,
lymphadenopathy
Diarrhea, vomiting, cachexia. Gastric
ulcers, esophageal ulcers,
and rectal masses (HIV patients)
Pleural effusion (HIV patients)
Fever, abdominal distension,
glomerulonephritis, and
renal amyloidosis
HIV, Plasmodium spp.,
M. tuberculosis, secondary
infections
Scaling, erosion, ulcers,
onychogryphosis, paronychia, cold
agglutinin disease (ear margin
necrosis). Nodules on mucous
membranes have also been
reported
Generalized lymphadenopathy
Diarrhea, melena, vomiting,
anorexia, weight loss, terminal
cachexia
Rhiniitis, epistaxis
Conjunctivitis, keratitis,
chorioretinitis, uveitis,
panophthalmitis
Asthenia, decreased activity,
glomerulonephritis, chronic active
hepatitis, polyarthritis, synovitis,
vasculitis
Rickettsia rickettsii, Ehrlichia canis,
E. platys, Babesia canis,
Demodex canis, Microsporum spp.
the human disease, and relapses are frequently observed.New alternative therapies are currently being investigated,including administration of cytokines, new compounds,and plant extracts (Table 6).37-41
Prophylaxis
The treatment of leishmaniasis in both humans and dogscan be unrewarding, emphasizing the need for additionalresearch and for prophylaxis. The prevention of leishmaniasis relies on a knowledge of the parasite and control ofthe vector. Complete eradication of mammalian reservoirhosts has been suggested and attempted, but is impractical
International Journal of Dermatology 1998, 37, 801-814
and, in the authors' opinion, would not eliminate the riskof leishmaniasis. It is probable that the eradication of thenatural host would result in the adaptation of the parasiteto different hosts, and the expansion of the species thatare currently using humans as reservoirs (L. donovani andserotypes of L. infantum). With regard to the role ofdogs in human leishmaniasis in the Mediterranean basin,euthanasia of seropositive and even affected dogs is notjustified as the prevalence of human infection in areasendemic for canine leishmaniasis is low and direct transmission of the disease from dogs to humans is only anecdotal.In addition, although HIV/Leishmania coinfection is clearlyan emerging problem, it has been shown that immunosup-
© 1998 Blackwell Science Ltd
Marsella and Ruiz de Gopegui
Table 4 Clinical pathologic abnormalities reported in humans and dogs with leishmaniasis
Leishmaniasis Review 811
Hematology
Chemistry
Humans
Non-regenerative anemia, increased ferritin"
Thrombocytopenia, thrombopathia
Hypoalbuminemia, hyperglobulinemia
Azotemia, increased liver enzymes
Dogs
'Non-regenerative anemia" (normochromic,
normocytic), increased ferritin, leukopenia,
plasma cells in peripheral blood samples
Thrombocytopenia, thrombopathia
Hypoalbuminemia, hyperglobulinemia
Azotemia, increased liver enzymes
Hematuria, proteinuria
Positive antinuclear antibody and Coomb's tests
*Anemia due to chronic inflammation.
Table 5 Diagnostic methods reported for human and canine leishmaniasis
Method Species Advantages Disadvantages
Parasitologic
diagnosis
Immunologic
diagnosis"
Cytology (Giemsa,
Romanowsky &
Wright, Diff-Quik)
Histopathology.
Immunoperoxidase
Culture (Novy-MacNeal
Nicolle or in Schneider's
culture medium)
PCR
IFAT
ELISA, dot-ELISA
Immunoblot
OAT
IPA
Recombinant L. infantum
proteins
Humans, dog
Humans, dog
Humans, dog
Humans, dog
Humans, dog
Humans, dog
Humans, dog
Humans, dog
Humans
Humans
High specificity, cheap
High sensitivity,
immunoperoxidase staining
improves the detection of
amastigotes
Sensitive, optimal for in vitro
studies evaluating sensitivity of
parasites to various drugs
High sensitivity
High sensitivity and specificity
Sensitive. Dot-ELISA improves
availability
Sensitive
Specific
Sensitive and specific
High specific
Low sensitivity,
interpretation
Time, availability
Results take 3-10 days,
expensive
Availability
Availability, interpretation
requires fluorescence
microscope, and less
sensitive than ELISA
Less precise than IFAT,
cross-reactions with Babesia
spp. and Trypanosoma
cruzi, and availability
Less sensitive than IFAT
*Immunologic determinations are optimal for epidemiologic studies, but do not confirm clinical disease. Immunosuppressedpatients may require parasitologic diagnosis as immune response may not be detectable. Parasitologic diagnosis is required inall cases of cutaneous disease, even in immunocompetent individuals.
pression is not the only factor in the epidemiology ofLeishmania infection in HIV-positive individuals, and thata more important factor is the sharing of infected syringes.P
Control of the vector is a more reliable strategy due to
© 1998 Blackwell Science Ltd
the fact that these insects spend their life cycle in a verylimited area (less than I km). Sand fly control can berestricted to inhabited areas only. The use of perrnethrintreated screens alone drastically reduced sand fly popula-
International Journal of Dermatology 1998,37,801-814
812 Review Leishmaniasis Marsella and Ruiz de Gopegui
Table 6 Therapeutic modalities used for the treatment of leishmaniasis in both humans and dogs
Group Compounds Administration Indications Mechanism of action Observations
Antimonials Meglumine Humans: 20-60 mgt New World Inhibits protozoa Contraindicated in
(pentavalent Sb antimoniate kg/day parenteral mucocutaneous carbohydrate and lipid pregnancy, cardiac,
compounds) Sodium (i.m.) leishmaniasis (L. metabolism hepatic, and renal
stilbogluconate Dogs: 100 mg/kg b.l.d, braziliensis) and disease
s.c. 15 days canine leishmaniasis
Antimicrobials Dapsone 200 mg/kg/day for 45 Old World cutaneous Exact mechanism is Hemolysis or
days leishmaniasis unknown. Thought to leukopenia may occur
decrease phospholipid
synthesis or to
interfere with folic acid
synthesis
Rifampicin (it can be 600-1200 mg/kg/day Old World cutaneous RNA-polymerase Contraindicated in
combined with leishmaniasis inhibitor pregnancy and liver
isoniazide) disease
Metronidazole 250 mg t.i.d. courses Old World cutaneous Cytotoxic effect on Contraindicated with
of 10 days/l0 days leishmaniasis parasite DNA ethanol. Efficacy
rest questioned
Trimethoprim- 160/800 mg b.i.d, 3-4 Old World cutaneous Folic acid metaboiism Efficacy questioned
sulfamethoxazole weeks leishmaniasis inhibitor
Paromoymycin Topically for 10 days, New World Interference with Combined with
plus antirnonials for 7 mucocutaneous protein synthesis antimonials for
days leishmaniases mucocutaneous
leishmaniasis
Aminosidine sulfate 16 mg/kg/day i.rn. Mucocutaneous Interference with
leishmaniasis protein synthesis
Xanthine oxidase Allopurinol 10-15 mg/kg b.Ld. New World Alters protein and Long-term
inhibitor mucocutaneous DNA synthesis of administration in
leishmaniasis and protozoa canine leishmaniasis,
canine leishmaniasis combined with initial
antimonial
administration
Antifungals Ketoconazole 200-400 mg/kg/day New World Ergosterol synthesis Side-effects include
mucocutaneous inhibitor anorexia, vomiting,
leishmaniasis (L. hepatitis, adrenal
mexicana) suppression, and
microsomal enzyme
suppression
Itraconazole 4 mg/kg/day (100-200 Old World cutaneous Ergosteroi synthesis Same side-effects as
mg/day) 6 weeks to 8 leishmaniansis inhibitor ketoconazole, but less
weeks hepatotoxic
Amphotericin B 0.5-1 mg/kg lipid New World Used in patients
complexes (liposomes) mucocutaneous unresponsive to
leishmaniasis and antimonials
visceral leishmaniasis
Physical Local heat 55°C for 5 min Old and New World Inhibition of Severe scarring may
cutaneous and amastigote replication occur
mucocutaneous
leishmaniasis
Cryotherapy End point is when an Old World cutaneous Severe scarring may
ice ball exceeding 2 leishmaniasis occur
mm over the margins
of the lesion is formed
International Journal of Dermatology 1998,37, 801-814 © 1998 Blackwell Science Ltd
Marsella and Ruiz de Gopegui
Table 6 Cont.
Leishmaniasis Review 813
Group
Cytokines
Compounds
Others
y-Interferon
Interleukin-12
Administration
Surgical excision,curettage, laser,electrodessication
Combined withconventional therapy
Combined withconventional therapy
Indications
Old World cutaneousleishmaniasis
Visceral leishmaniasis(experimental)
Visceral leishmaniasis
(experimental)
Mechanism of action Observations
Increases macrophage Experimentalnitric oxide production
Modulates T-cell Experimentalresponse (inhibits Th2cell IL-4 production)
tions in one preliminary study.O In addition, plants thatinhibit the development of Leishmania in sand flies andBacillus sphericus infection of Phelbotomus have beeninvestigated.v-t"
The development of a vaccine against leishmaniasis is along-term goal in both human and veterinary medicine.Protective immunity occurs in the majority of humanpatients with cutaneous leishmaniasis after the resolutionof the clinical signs, and this is the reason why someprimitive populations inoculated their children withinfected materials. I Immunization has been attempted onan experimental basis with killed parasites, parasite fractions, and genetically engineered organisms, including eithermodified Leishmania or Salmonella.ri At the present time,however, a vaccine is not commercially available.
Conclusions
Leishmaniasis remains a serious public health problem inmany regions of the world. Long-term goals to improvethe control of this disease include the development of avaccine and safer drugs that can be administered orallyand for a shorter period of time.
Drug names
Allopurinol, Zyloprim; aminosidine and paromomycin sulfate (not available in the USA); amphotericin B, Fungizone;dapsone, Dapsone Tablets; y-interferon, Actimmune; itraconazole, sporonox; ketoconazole, Nizoral; rifampicin,Rifadin; trimethoprim-sulfamethoxazole, Bactrim.
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