World Association for Veterinary Dermatology Consensus Statement for Diagnosis, and Evidence-Based Clinical Practice Guidelines for Treatment and Prevention of Canine Leishmaniosis

World
Association for Veterinary Dermatology consensus statement for diagnosis, and
evidence-based clinical practice guidelines for treatment and prevention of
canine leishmaniosis

Manolis
N. Saridomichelakis*, Gad Baneth^†, Silvia
Colombo^‡, Filipe Dantas-Torres^§, Lluís
Ferrer^¶, Alessandra Fondati**, Guadalupe Miró^††, Laura Ordeix^¶, Domenico Otranto^‡‡, Chiara Noli^§§

* Clinic of Medicine, Faculty of Veterinary Science, University of
Thessaly, Trikalon Str. 224, GR-43132, Karditsa, Greece

^† Koret School of
Veterinary Medicine, Hebrew University of Jerusalem, PO Box 12, Rehovot
7610001, Israel

^‡
Studio Dermatologico Veterinario,
Milano, Italy; Clinica Veterinaria
Nervianese, Nerviano,
Milano, Italy

^§
Magalhães Institute, Fundação
Oswaldo Cruz (Fiocruz), Recife, Brazil

^¶
de Medicina i Cirurgia Animals, Fundació
Hospital Clínic Veterinari,
Universitat Autònoma de
Barcelona, 08193 Bellaterra, Spain

** <spana
M.C. Cetego 20, 00177, Roma, Italy

^††<spanl
Health, Veterinary teaching Hospital, Faculty of Veterinary, Universidad
Complutense de Madrid, Madrid, Spain

^‡‡
Department of Veterinary Medicine, University of Bari, Valenzano, Bari, Italy;
Department of Veterinary Clinical Sciences, City University of Hong Kong, SAR,
China

^§§ Servizi Dermatologici Veterinari, Strada
Bedale della Ressia 2, Peveragno (CN), 12016, Italy

Correspondence: Manolis N. Saridomichelakis, Clinic of
Medicine, Faculty of Veterinary Science, University of Thessaly, Trikalon Str.
224, GR-43132, Karditsa, Greece. E-mail: msarido@vet.uth.gr

Conflict of Interest Statement: during the last 5
years the authors have received research support, lecture honorarium and/or
consultation fees from the following commercial companies: MNS: Bayer, Ceva,
Elanco, Hellafarm, MP Labo, MSD, Premier Shukuroglou, Provet, Virbac; GB:
Bayer, Elanco, MSD, Virbac; SC: Elanco, Zoetis; FDT: Bayer, Elanco, Labyes,
MSD, Vetoquinol; LF: Affinity Petcare, Bioibérica, Ceva, Elanco, IDEXX, LETI, Zoetis;
AF: Affinity Petcare, Ecuphar, Dechra, MSD, Vetoquinol, Zoetis; GM: Bayer,
CEVA, Elanco, Letipharma, MSD; LO: Affinity Petcare, Bioibérica, Dechra,
Elanco, Hill’s, Hipra, Purina, Vetoquinol, Virbac, Zoetis; DO: Bayer, Boehringer
Ingelheim, Ceva, Elanco, MSD, Virbac, Zoetis; CN: Aurora Biofarma, Ceva,
Dechra, Elanco, Hill’s, Innovet, MSD, Purina, Royal Canin, Vétoquinol, Zoetis,
Farmina, Nextmune.

Sources of Funding: World Association for
Veterinary Dermatology

Acknowledgements: authors want to
express their gratitude to Dr Francesca Abramo for providing histopathological
images

Previous Presentations: the evidence-based clinical practice guidelines
for treatment and prevention of canine leishmaniosis were presented by the last
author, in an invited lecture, at the 10^th World Congress of
Veterinary Dermatology, Boston, MA, U.S.A., July 2024

Running Title: Guidelines for
canine leishmaniosis

ORCID iD

Manolis N. Saridomichelakis: 0000-0001-8308-3371

Gad Baneth: 0000-0002-7549-1305

Silvia Colombo: 0000-0003-1138-4364

Filipe Dantas-Torres: 0002-1496-6274

Lluís Ferrer: 0000-0003-1537-0074

Alessandra Fondati: 0009-0002-2202-214X

Guadalupe Miró: 0000-0003-0981-2470

Laura Ordeix: 0000-0002-3631-8358

Domenico Otranto: 0000-0002-7518-476X

Chiara Noli: 0000-0002-2310-9813

Author
Contributions:

Manolis N. Saridomichelakis: data curation (lead), formal analysis (lead), investigation
(lead), methodology (lead), writing-original draft (lead), writing-review and
editing (lead); Gad Baneth: writing-original draft (equal); writing-review and editing
(equal); Silvia Colombo: writing-original draft (equal), writing-review and editing
(equal); Filipe Dantas Torres: writing-original draft (equal), writing-review and editing
(equal); Lluis Ferrer: writing-original draft (equal), writing-review and editing
(equal); Alessandra Fondati: writing-original draft (equal), writing-review and editing
(equal); Guadalupe Miró: writing-original draft (equal); writing-review and editing
(equal); Laura Ordeix: investigation (supporting),
writing-original draft (equal), writing-review
and editing (equal); Domenico Otranto: writing-original draft (equal), writing-review and editing
(equal); Chiara Noli: conceptualization,
data curation, formal analysis, funding acquisition, investigation,
methodology, project administration, supervision, validation, writing-original
draft (equal), writing-review
and editing (equal)

Abstract

Background – Canine leishmaniosis (CanL)
due to Leishmania infantum remains common, and veterinarians do not always follow scientifically sound approaches for
diagnosis, treatment and prevention.

Objectives – To provide consensus guidelines for diagnosis and evidence-based
guidelines for treatment and prevention of CanL.

Methods and
material – Clinical consensus guidelines for the
diagnosis were structured based on literature and authors’ experience. Three
electronic databases were searched for randomized controlled trials, systematic
reviews and metanalyses on treatment and prevention.

Results,
conclusions and clinical importance – Diagnosis
should be based on compatible clinical signs and/or clinicopathologic
abnormalities, exclusion of differentials, demonstration of infection and
increased concentration of anti-Leishmania IgG (quantitative serology). Euthanasia for public health purposes is not
recommended and drugs with anti-Leishmania activity should be avoided in
subclinically infected dogs. Recommended treatments include meglumine antimoniate-allopurinol
(first line treatment), miltefosine-allopurinol (first line treatment) and aminosidine-allopurinol
(second line treatment); marbofloxacin may be considered in dogs with advanced
chronic kidney disease. In endemic areas, recommended measures for prevention
include deltamethrin 4% collar, flumethrin 4.5%-imidacloprid 10% collar or
permethrin 50%-imidacloprid 10% spot-on, regular administration of isoxazolines,
not using infected blood products for transfusion, not breeding seropositive bitches
or dogs with CanL, administration of domperidone (seronegative dogs) and
dietary nucleotides-active hexose correlated compound (subclinically infected,
seropositive dogs). Vaccination with LiESP with MDP may be considered,
whereas protein Q vaccine is recommended in areas with very high rates of
seroconversion. In non-endemic
areas, recommended measures include <spand
removal of infected female dogs from reproduction.

Keywords: allopurinol;
aminosidine; deltamethrin; domperidone; flumethrin; meglumine antimoniate;
miltefosine; nutritional supplement; permethrin; vaccine

1. Introduction

1.1. Etiology

            The genus Leishmania includes
kinetoplastid protozoa transmitted by phlebotomine sand fly vectors of the
genus Lutzomyia (subdivided into different genera in a recent taxonomic
revision) and Phlebotomus, in the New and Old World respectively. These
protozoa are the causative agents of leishmaniases, which are diseases with different degrees of severity, affecting several animal species and humans, in
most continents.¹ Leishmania infantum is usually associated
with visceral leishmaniasis (VL) but may also cause cutaneous lesions [cutaneous
leishmaniasis (CL)], which are more typical of other dermotropic species of the
genus (e.g., L. major, L. tropica). Furthermore, some of the
latter may invade internal organs (e.g., L. amazonensis), and some other
species can cause mucocutaneous disease (e.g., L. braziliensis). These
protozoa are included in the subgenera Leishmania and Viannia according
to the localization of developmental stages in the digestive tract of their
sand fly vectors and to their biochemical and molecular characteristics,
whereas Leishmania species of lizards are included in the subgenus Sauroleishmania.
The characterization of Leishmania species and strains should rely on
the isolation of the parasite in culture followed by multilocus enzyme
electrophoresis or another reference method, such as DNA sequencing, and on the
molecular characterization of specific targets [e.g., kinetoplast DNA (kDNA),
intergenic transcribed spacer region-1 (ITS-1), heat shock protein 70 (hsp70)
gene], according to the aims and the required level of parasite identification.

            Leishmaniases are listed amongst the neglected tropical diseases greatly
impacting, in terms of morbidity and mortality, human populations worldwide
with a yearly burden of approximately 0.2 to 0.4 million cases of VL and of 0.7
to 1.2 million cases of CL.¹ Human VL is mostly prevalent in
developing countries with more than 90% of cases reported in Bangladesh,
Brazil, Ethiopia, India, South Sudan, and Sudan. Similarly, 70-75% of CL cases
are reported in Afghanistan, Algeria, Brazil, Colombia, Costa Rica, Ethiopia,
Iran, North Sudan, Peru, and Syria. Nonetheless, these diseases are often
underreported or not diagnosed at all, in remote rural areas of the world,
suggesting that the figures above are probably underestimations.

            Like in humans, multiple Leishmania species infect dogs,
potentially leading to diseases with variable clinical and clinicopathological
manifestations. Among them, L. infantum is the most important one from a
global perspective and can cause a disease characterized by both cutaneous and
visceral involvement, called canine leishmaniosis (CanL). Unless otherwise
stated, the remaining of this article will be devoted to CanL due to L.
infantum.

1.2.
Epidemiology

            Leishmaniosis
caused by L. infantum is probably the
most important canine vector-borne disease of zoonotic concern, being prevalent
all over the world except Oceania.^2,3 The infection is
distributed, in relationship to the presence of sand fly vectors, in Far East
Asia, Africa, Middle East, Europe, and Central and South America.⁴ Genetic studies suggest that L.
infantum was introduced to the New World by the Conquistadores via infected
dogs,⁵ and then the protozoon found suitable vertebrate hosts and proper sand
fly vectors to perpetuate. In
the last decades, the distribution of CanL has expanded in many geographic
areas where it was not previously endemic, such as northern Argentina,⁶ United States, as well as in northern regions of Italy and Spain.^7-9 The reasons for this expansion are mainly linked to the fact that sand
fly vectors are colonizing new ecological niches due to global increase in mean
temperatures, the human and animal movements and other human activities (e.g.,
deforestation, urbanization, etc.).^2,10,11 Also, the lack or
inefficacy of control programs (e.g., diagnosis, monitoring, and use of
appropriate repellents in dogs) as well as the occurrence of reservoir animals
other than dogs may represent important factors favoring the presence of new
foci of infection.¹²

            New cases of canine infection occur when sand flies are active, that is
throughout the year in the New World and from spring to autumn (i.e.,
from May to October) in the Old World, although there are some endemic areas in
Europe, such as Southern Spain, where the transmission season has been expanded
to almost 10 months per year.⁸ Sand flies are small, fragile
insects that may virtually colonize almost all environments, from
forests to human houses, and from coastal plains to hilly areas, if proper
conditions to complete their biological life cycle (e.g., habitat with organic
matter, high humidity, etc.) are available.⁴ Another important component in the natural transmission chain of L.
infantum is represented by the presence of potential vertebrate hosts,
other than dogs, in different ecotypes.¹³ Although dogs are the main
peridomestic reservoir of L. infantum
worldwide, the parasite has been isolated from many other classes of mammals
(e.g., rodents, lagomorphs, marsupials, non-human primates, and carnivores).^2,14 The role played by these animal
species as reservoirs of L. infantum depends on a complex
chain of factors in different ecological
contexts (e.g., sand fly vector
density, species composition and blood feeding preferences, length of
transmission season).

            Dogs and cats have been shown to act
as a source of L. infantum infection for phlebotomine sand flies, whereas
studies about other animal species are scant and their possible role is usually
inferred by the delineation of the blood source of infected engorged female sand
flies. However, both the black rat (Rattus rattus) and the Iberian hare
(Lepus granatensis) have been demonstrated to infect sand flies.^15,16 A
paradigmatic example is represented by the Iberian hare, which has been
implicated in the most important outbreak of human leishmaniosis (2009-2012)¹⁷ known in the history of Europe, in people frequenting a suburban park
near Madrid, Spain.¹⁸ The prevalence of seropositivity was not increased in dogs from the
same area, and conversely, up to 45% of hares sampled were infected by L.
infantum. In addition, naturally-infected hares were infectious to Phlebotomus
perniciosus, and this sand fly species showed a high preference to feed
from hares under natural conditions. Based on some experiments of sand fly
feeding preferences and on the molecular detection of L. infantum in red
foxes (Vulpes vulpes), this animal
has also been suggested as a putative reservoir of L. infantum.^19,20 A plethora of
other animals such as opossums and wild canids have also been regarded as
potential reservoirs of L. infantum in
Latin America.^21,22 From a public health perspective, the presence of reservoirs other than
dogs could reduce the effectiveness of control programs based on the
application of repellents on dogs.

The dynamic of L. infantum infection in dogs is multifactorial
and it is linked to the vectors (e.g., species composition, density, host
preference, length of transmission season) and the presence and availability of
infected reservoir hosts, primarily other dogs.²³ In endemic areas, dogs may remain subclinically infected (i.e., not
presenting clinical signs or clinicopathologic abnormalities of CanL) for all
their life,²³ but their infectivity increases when they develop CanL.²⁴

2. Pathogenesis and immunology of
canine leishmaniosis

            In the vertebrate host,
amastigote (non-flagellated) forms of Leishmania spp. replicate inside
macrophages, which are ingested by the sand fly vectors along with their blood
meal. Subsequently, they transform to promastigotes and replicate in the gut of
the insect until the flagellated metacyclic promastigotes are inoculated to a
new receptive host.⁴ However, blood transfusion,
direct, and vertical or venereal transmissions have also been demonstrated as
alternative modes of infection, that are particularly important in areas where
suitable phlebotomine sand fly vectors are not present.^25,26

            Infection is initiated when the female sand fly introduces into the
superficial dermis of the dog the metacyclic promastigotes that are lodged in
its intestinal tract, together with saliva, intestinal microbiota, and other
products, such as promastigote secretory gel (PSG), which play a determining
role in the establishment of infection. For example, saliva acts by inhibiting
hemostasis, whereas the bacteria of sand fly intestine and PSG act as
pro-inflammatory factors.²⁷ In the next few hours, an acute
inflammatory response is triggered at the inoculation site, and neutrophils are
the first cells to arrive, attracted by cytokines and chemokines such as IL-1β
and CXCL1. After neutrophils, the next cells to become infected are the
resident and inflammatory dermal macrophages. Notably, apoptotic parasitized
neutrophils are phagocytosed by macrophages, contributing to their high infection
rates, in a “Trojan Horse” model of infection.²⁷ Then, infected macrophages migrate to the regional lymph nodes, where
they initiate the adaptive immune response, and they enter the blood stream
(parasitemia) circulating and homing to different internal organs.²⁸ Bone marrow, spleen and lymph nodes are usually among the first,
although, as the disease progresses, most, if not all, parenchymal organs may
become infected. In dogs, L. infantum has a marked dermotropism and the
skin is one of the main target organs. Therefore, except the site of
inoculation, numerous additional areas of skin infection develop after
hematogenous spread, with a multifocal or generalized distribution. The
dissemination of the infection to the skin is essential for the transmission of
parasites to the sand fly vector and is responsible for most of the skin
lesions.²⁹

            The immune responses mounted by the canine host play
an important role in the susceptibility to L. infantum and development
of disease.³⁰ Interferon-γ activates macrophages to kill intracellular amastigotes
through the production of reactive oxygen species, and this has been shown to
be a protective cell-mediated immune pathway, which enables the control of
infection. Conversely, immune responses that induce the secretion of
interleukin-4 and the evolution of B-cells into plasma cells with increased
immunoglobulin production are linked to uncontrolled infection and progression
to CanL. Research in
rodents infected by dermotropic Leishmania species, such as L. major,
has shown that the T-helper 1 (Th1) type of immune
response with its associated cytokine cascade led to parasite elimination by
activated macrophages and resistance. On the contrary, the T-helper 2 (Th2)
immune response led to increased parasite load and production of non-protective
anti-Leishmania antibodies and finally to disease. Dogs usually develop
mixed Th1/Th2 responses, and the
balance between them determines the course of infection.^28,30

            The progression from infection to
CanL is marked by a depressed cell-mediated immunity and an extreme
upregulation of humoral response. In chronic CanL, dogs increasingly express
the programmed death-1 (PD-1) cell-surface receptor on their lymphocytes (T
cell exhaustion) and experience diminished lymphocyte proliferation responses
upon stimulation, initially with L. infantum antigen (parasite-specific
suppression of cell-mediated immunity) and later with irrelevant mitogens
(generalized suppression of cell-mediated immunity).^28,31,32 At
the same time, circulating
immune complexes are formed and their deposition in special vascular plexuses
mediate some important pathological manifestations of CanL,³³ such as glomerulonephritis, uveitis,
arthritis, and vasculitis.^28,34-36 In addition,
the presence of parasites triggers macrophagic and lymphoplasmacytic
inflammation in multiple organs.

            Susceptibility or resistance to CanL are also influenced by the dog’s
genetic makeup. Severe CanL is rare among Ibizan hounds in the Balearic islands
of Spain and its prevalence is significantly less common compared to other
canine breeds in the same L. infantum-endemic islands.³⁷ It has been shown that the Ibizan hound produces a predominantly
cellular response against L. infantum while other breeds, that
evolved in non-endemic areas, such as the Boxer, Rottweiler, and German
shepherd dogs, are more susceptible and are overrepresented in CanL surveys.^38,39

3. Non-cutaneous manifestations
of canine leishmaniosis

            The clinical manifestations of
CanL are broad and variable among dogs, mainly due to the differences in their
immune responses and the multiplicity of pathogenic mechanisms.²⁸ In general, CanL is a chronic, multisystemic disease that may affect almost every
system and organ, with severity varying from mild and self-limiting to fatal.³⁰

3.1. History and common clinical manifestations

            The typical history reported by
owners of dogs with CanL includes the appearance of skin lesions, ocular
abnormalities, weight loss, lethargy, exercise intolerance, lameness, and
epistaxis. Dogs with chronic kidney disease (CKD) or other internal organ
involvement (e.g., liver, gastrointestinal, respiratory system) may be admitted
due to additional symptoms such as polyuria/polydipsia (PU/PD), anorexia,
vomiting, diarrhea, melena, or sneezing.⁴⁰

            On
physical examination, the main non-cutaneous findings associated with CanL are
peripheral lymphadenomegaly, pale mucous membranes, splenomegaly, ocular
lesions, poor body condition, muscle atrophy involving mainly the masticatory
muscles, rhinitis, and joint swelling.^30,41 In particular, the prevalence
of ocular lesions such as keratoconjunctivitis and uveitis varies from 12% to
71% in different canine populations and studies.^42-44

            When muscle atrophy affects
mainly the temporal muscles it is attributed to chronic masticatory muscle
myositis, whereas it is generalized in cachectic animals.^41,45 Polymyositis has also been
described is some cases.⁴⁶

            Gastrointestinal
manifestations may appear in conjunction with other clinical signs of CanL and
more rarely as the only clinical presentation, especially in certain breeds
such as the Boxer and German shepherd dog.⁴⁷ They include small intestinal
diarrhea, with or without melena, and symptoms due to ulcerative granulomatous
colitis.^48,49 Ascites and vomiting due to
liver disease are rare.⁴⁰

            Central
nervous system (CNS) inflammation, usually manifested by clinical signs of encephalitis, has been described
in CanL.^50,51 Some inflammatory lesions are
characterized by the presence of abundant T lymphocytes and mononuclear cells
and may be due to co-infections with pathogens such as Toxoplasma gondii and
Neospora caninum.^52,53 In other cases, damage of CNS
vascular bed leads to infarctions.⁵⁴ Blood-brain barrier compromise
has been demonstrated and may explain high levels of anti-L. infantum IgG in the cerebrospinal fluid.^55,56 However, L. infantum has also
been detected in the CNS of dogs with CanL but no neurological signs, which implies
that the mere presence of the parasite does not necessarily mean that it is
responsible for these signs.^57,58

            The
respiratory system may also be affected. Chronic rhinitis is common,⁵⁹ whereas chronic interstitial
pneumonia has been detected histopathologically but without associated clinical
signs.⁶⁰

            Although
rare, some dogs with CanL and heart disease have been described, with evidence
of myocarditis and local presence of the parasite.^61,62

            The
disease may also affect the male and female reproductive system. Males may
present low semen quality with reduced progressive motility and increased
number of spermatozoa with morphological abnormalities. Semen quality appears
to be partially restored after long-term allopurinol administration.⁶³ In females with CanL placentitis
due to L. infantum has been described after abortion.⁶⁴

3.2. Clinicopathologic
abnormalities

            About 63% of the dogs admitted
with CanL are anemic, usually with mild-to-moderate non-regenerative anemia,
and 25% have lymphopenia.³⁶ Normocytic-normochromic
non-regenerative anemia develops as in other chronic debilitating inflammatory
diseases which affect hematopoiesis and worsens when CKD develops. Hemorrhage
and hemolysis may contribute to anemia in some dogs. Mild to moderate
thrombocytopenia is common. Anti-platelet antibodies are present in some dogs
with CanL, opsonizing the thrombocytes and decreasing their lifespan in the
circulation. In addition, the decreased clotting capacity of platelets
(thrombocytopathy), along with vasculitis and serum hyperviscosity, can result
in bleeding tendency, which, in combination with ulcerative rhinitis, explains
epistaxis.^65,66

            <spanm
biochemistry findings are hyperproteinemia, hyperglobulinemia (mainly due to
the increased antibody production) and hypoalbuminemia (due to glomerular loss
and inflammation).^{30,<span; ‘>36,<span; ‘>67</span;>}<spane
phase proteins, such as C-reactive protein (CRP)
and ferritin, are increased whereas not only albumins but also paraoxonase 1
(PON1), another negative acute phase protein, is decreased.⁶⁸
Exceptionally elevated activities of liver enzymes are found in a minority of
dogs, whereas azotemia, proteinuria [increased urine protein/creatinine ratio (UPC)]
and low urine specific gravity may be found in cases with CKD, mainly due to immune complex deposition on the glomeruli.³⁴. Proteinuria, that may initially be
reversible, is often present long before CKD deteriorates enough to result in
increased blood creatinine, symmetric dimethylarginine (SDMA), urea and
inorganic phosphorous concentrations, at which stage the prognosis starts
becoming poor. Indeed, CKD is considered the main cause of natural death in
CanL.^23,6970-72

            The skin lesions in CanL are
characterized by extreme pleomorphism, variable severity, and are uncommonly
associated with pruritus. The clinical pleomorphism is reflected in the
histopathological features as well. It is partially unknown why the disease can
have so many different cutaneous presentations, however the interaction between
the host immune system and the parasite is suspected to play a role in this.^70-72

            Cutaneous manifestations of CanL can
be divided into typical and atypical. Typical clinical patterns are highly
suggestive of CanL, and include exfoliative (scaling) dermatitis, ulcerative dermatitis
affecting bony prominences, papular dermatitis and onychogryphosis. Atypical
clinical patterns are less specific and less suggestive of CanL and may mimic
many other diseases. These include pustular dermatitis, nodular dermatitis,
ulcerative dermatitis other than ulcers on bony prominences, and footpad and/or
nasal hyperkeratosis.^71,72 Affected dogs
may present with a single clinical pattern or more than one. Clinical patterns
of CanL are the same in endemic and non-endemic areas, however prevalence of
the different presentations may vary.⁷³

            Differential diagnoses for each
clinical pattern and useful clinical hints (“clinical pearls”) to help the
clinician are summarized in Table 4.1. Clinical pearls are defined as practical
medical tips based on experience and personal observations.⁷⁴ The most important determinant of validity of a clinical pearl is the
number of observations: the more numerous the observations, the greater their
diagnostic value. However, considering that most pearls are personal opinions
and not evidence-based data, they should be used with caution.

4.1.
Exfoliative dermatitis

            Exfoliative (scaling) dermatitis
(Figure 4.1) is the most common dermatological presentation of CanL, and its
prevalence has been reported to be between 45.7% and 98.7%.⁷⁰ It is characterized by large, dry, whitish scales, often described as
asbestos-like, variably adherent to the underlying skin. When scales are very
adherent, they may be more easily palpated than seen, and their removal can
leave an erosion. Along with the scales, follicular casts, partial alopecia, or
both may be present. Pruritus is usually absent unless there is secondary
bacterial infection. Lesions may initially involve the face and ear pinnae,
with a symmetrical distribution around the eyes, and then extend to the hairy
surface of the trunk and limbs. Distribution may be localized, regional or generalized,
and symmetrical or asymmetrical.^70-72

Histopathologically, this pattern is characterized by epidermal and
follicular orthokeratotic hyperkeratosis and a perivascular to interstitial
infiltrate in the superficial and mid dermis (Figure 4.2), with or without
inflammation and destruction of the sebaceous glands (sebaceous adenitis). The
dermal infiltrate may also be more intense, from nodular to diffuse, and may
involve the panniculus (pyogranulomatous to granulomatous panniculitis). The
predominating inflammatory cells are macrophages, plasma cells and lymphocytes;
less commonly neutrophils, eosinophils, and mast cells are found, whereas
multinucleated giant cells are rare. Variable numbers of amastigotes may be
identified in biopsies of dogs presenting with exfoliative dermatitis.^75,76

Idiopathic sebaceous adenitis is the main differential when follicular
casts are observed during clinical examination. Histopathologically,
involvement of the sebaceous glands in the inflammatory process can be observed
in half of the skin biopsies obtained from areas with exfoliative dermatitis.^70,76 Sebaceous
adenitis associated with CanL is characterized by a multinodular to diffuse
dermal inflammatory infiltrate not limited to the perifollicular dermis.⁷⁷ It has been suggested that when no inflammatory infiltrate is observed
in the dermis but it is strictly centered to the sebaceous glands, idiopathic
sebaceous adenitis is more likely than CanL, even in endemic areas.⁷⁷

4.2.
Ulcerative dermatitis

            Ulcerative dermatitis is the second
most frequent cutaneous manifestation of CanL after exfoliative dermatitis.⁷⁰ The ulcerative lesions of CanL are commonly grouped together, despite
different clinical presentations being observed, reasonably suggestive of a
different underlying pathogenesis. Clinical patterns include ulcers i) on sites
subjected to trauma, like bony prominences-pressure points and sites of
preexisting wounds, ii) on body extremities (paws, apex of ear pinnae, nose,
tip of tail), and iii) on nasal planum and/or mucocutaneous junctions. In all
cases they are non-pruritic, variably painful, chronic ulcers, that frequently
do not respond to antibiotics. Ulcers on bony prominences, unlike other
ulcerative patterns, represent a relatively striking clinical presentation of
CanL; the remaining ulcerative patterns are more likely to overlap, both
clinically and histologically, with other diseases.⁷²

4.2.1. Ulcerative
dermatitis on sites subjected to trauma

            a)
On bony prominences-pressure points (Figure 4.3): they appear as chronic,
indolent, deep ulcers with sharp raised borders usually affecting carpal and
tarsal joints, or the ischiatic tuberosities. They can be solitary or multiple,
and unilateral or bilateral. It has been hypothesized that continued pressure
causes secondary inflammation with subsequent infiltration of infected
macrophages, that results in more severe inflammation and ulcer development.⁷⁰

b) On the site
of pre-existing wounds: less frequently, persistent ulcers have been reported
at sites of pre-existing injury with loss of skin integrity, like in surgical wounds,⁷⁸ or in lesions of acral lick dermatitis (Figure 4.4).⁷⁹ Ulcerated nodules and plaques may also develop in some cases. It has
been hypothesized that these lesions are the consequence of the influx of
infected macrophages at the site of trauma during the normal wound healing
process. The extracellular release of parasites perpetuates inflammation,
interferes with healing, and explains the chronicity of the lesions. A similar
pathogenetic mechanism has also been described in humans who have been
presumably infected while travelling to endemic areas and, within weeks to
months after their return, they develop CL with ulcers at the site of even
minor mechanical trauma, such as insect bites, tattoos, or shaving cuts.^80,81 The peculiarity of these lesions is that the initial injury causing disruption
of skin integrity is irrelevant to the parasite.⁸²

            Dermatopathological
examination of biopsies taken from ulcers at sites subjected to trauma often
shows epidermal hyperplasia with ulceration and a periadnexal to diffuse
neutrophilic-macrophagic dermal infiltrate with variable number of amastigotes.^70,72

4.2.2. Ulcerative dermatitis
on body extremities

            In this case, the pathogenesis of the ulcers is attributed to cutaneous
vasculitis with deposition of circulating immune complexes in the vessel wall.
The characteristic lesions consist of occasionally bleeding ulcers covered by
hemorrhagic crusts, typically located on the margins of the pinnae, and less
commonly on the tip of the tail, digits, and paw pads (Figure 4.5).
Occasionally, onychomadesis with subsequent onychodystrophy can be observed,
resulting from vascular damage to the nail matrix.⁷² Infrequently, in addition to or in lieu of lesions indicative of
vasculitis, signs of ischemic dermatopathy can be present, such as multifocal
alopecic areas characterized by cutaneous atrophy, scaling, hypo- or
hyper-pigmentation distributed mainly on the head and distal legs.⁷²

Although the type and distribution of these lesions strongly suggest
vascular damage, this is rarely documented because they are not frequently
subjected to dermatopathological examination, mainly because of their location
that makes difficult to perform skin biopsies. In addition, vascular
inflammation may be temporary and does not affect all vessels, making it very
complicated to document it. However, even if vascular damage is confirmed histologically,
it would still be difficult to attribute a causal role to the parasite. Its intralesional
presence may not be demonstrable, even by molecular techniques, since these
lesions are induced by the deposition of circulating immune complexes. The
diagnosis of leishmaniosis in these cases is mostly based on the remaining
clinical signs, suggestive clinicopathologic changes, exclusion of
differentials, demonstration of the infection and of elevated levels of
circulating anti-Leishmania antibodies.³³

4.2.3. Ulcerative dermatitis of
nasal planum and/or mucocutaneous junctions

            Nasal planum dermatitis caused by CanL is characterized by erosions,
ulcers, crusts, scales, and variable depigmentation, with possible swelling and
loss of the typical cobblestone architecture (Figure 4.6).^72,83 These lesions can also involve the nasal orifices and alar folds and
may be accompanied by scales and crusts on the hairy skin of the dorsum of the
nose. Discoid lupus erythematosus (DLE) is the main differential, both
clinically and histologically.⁸³ A retrospective study comparing histopathological and
immunopathological features of nasal planum dermatitis in 20 dogs with DLE or
CanL showed a band-like lymphoplasmacytic dermatitis at the dermo-epidermal
junction, with basal cell vacuolation and occasional apoptosis in both
diseases. However, a nodular-to-diffuse superficial and/or deep infiltrate,
composed of macrophages, lymphocytes, and plasma cells, was visible only in CanL.⁸³ Amastigotes were not seen in any of the hematoxylin-eosin (H&E) stained
sections, denoting that the number of parasites is low. CD20-positive
lymphocytes predominated over CD3-positive T cells in both diseases, but the
percentage of dermal Mac387-positive macrophages was significantly higher in
CanL compared to DLE.⁸³

Variably depigmented, non-pruritic, scaling, crusting, erosive and
ulcerative lesions of other mucocutaneous junctions may also be seen in CanL.⁷¹ All mucocutaneous junctions can be affected; however, in addition to
the nasal planum, the medial canthus of the eyes and lips appear to be more
frequently involved (Figure 4.7). Histopathological findings of mucocutaneous
lesions in dogs with leishmaniosis caused by L. infantum have not been
reported, however they are likely to resemble those observed in mucocutaneous
pyoderma and characterized by a perivascular to interstitial to band-like,
predominantly plasmacytic infiltrate at the dermo-epidermal junction,
accompanied by lymphocytes, macrophages, and neutrophils.⁷² Mucocutaneous pyoderma histologically overlaps not only with the
mucocutaneous lesions of CanL, but also with both chronic DLE and mucocutaneous
lupus erythematosus, that can also be accompanied by bacterial superinfection.^84,85 Therefore,
whenever mucocutaneous pyoderma is included in the diagnostic hypotheses,
specific antimicrobial treatment prior to biopsy is recommended.^72,85

4.3. Papular dermatitis

            In endemic regions, this is a very characteristic primary cutaneous
manifestation of L. infantum infection. Although the exact prevalence is
unknown, it is very common (54%) in resistant breeds, such as the Ibizan hound.⁸⁶ Lesions start as raised erythematous papules in sparsely haired skin,
such as the inner pinnae, eyelids, dorsal part of the nose, lips, caudal
abdomen, and medial thighs (Figure 4.8). Sometimes, the papules tend to
coalesce to reach a final size of a small plaque. A crust develops in the
center of each papule, covering an ulcer with a raised edge and umbilicated
appearance.⁷¹ This clinical appearance is similar to the one observed in the
localized form of human CL, known as the “volcano sign”. It is highly suspected
that papules develop at the site of Leishmania inoculation in dogs with
strong cell-mediated immunity against L. infantum.⁷¹ Evidence for this hypothesis is the lesion distribution, reduced
parasite dissemination to internal organs, low parasite load in lesional skin,
lack of other clinicopathological abnormalities, low or negative specific
antibody levels, positive results of the leishmanin
skin test (LST), high expression of IFN-g in blood and
lesional skin, and spontaneous resolution over 3-5 months.^87-90 The histopathological picture of papular dermatitis is dominated by a
nodular to diffuse granulomatous dermatitis, without multinucleated giant cells
and usually with few parasites (Figure 4.9).⁸⁹

4.4. Cutaneous and
mucocutaneous nodular dermatitis

            Cutaneous and mucocutaneous nodular dermatitis is a relatively uncommon clinical
presentation, with a prevalence of up to 12%.⁷⁰ It is described more frequently in the Boxer breed. Clinically, it is
characterized by single or multiple plaques or nodules of variable size (1-10
cm), usually located on the head, distal limbs, and thorax (Figure 4.10).
Unlike papular dermatitis, these lesions are localized on haired areas and
sometimes they ulcerate. In some cases, these lesions have been described on
mucocutaneous junctions and mucous membranes, such as the oral or genital
mucosa.⁹¹

            The nodules are attributed to the
spread of the parasite to the skin or mucous membranes via the
lympho-hematogenous pathway in moderately to severely affected dogs.
Dermatopathological examination reveals a diffuse granulomatous or
pyogranulomatous dermatitis, sometimes with multinucleated giant cells and with
a variable but frequently high number of amastigotes.^92,93

4.5. Pustular dermatitis

            Pustular dermatitis is an uncommon presentation of CanL, with a
prevalence ranging between 1 and 13% of the cases.⁷⁰ Typical lesions are variable-sized pustules, surrounded by an
erythematous halo and admixed with erythematous papules, epidermal collarettes
and crusts, all representing various stages of lesion evolution (Figure 4.11).
Lesions may show a polycyclic or arciform configuration and multifocal alopecia
may occasionally be present. Pustules show generalized, symmetrical
distribution all over the body, involving both densely and sparsely haired
areas. Pruritus is variable, and often severe.^71,72,94,95 Affected dogs
may also show systemic signs such as anorexia and fever.

            A recent study showed that in
endemic areas there is a statistically significant association between CanL and
this uncommon clinical presentation,⁹⁵ but their etiopathogenetic relationship remains unclear. Two hypotheses
have been proposed: i) an immune-mediated pustular dermatitis develops in a dog
infected by L. infantum and the ensuing dysregulation of the immune system
causes the infection to progress to CanL, and ii) the immune-mediated pustular
dermatitis may occur secondary to CanL-induced immunologic abnormalities.
Moreover, in some cases, the possibility of an adverse drug reaction as
underlying cause of pustular dermatitis cannot be ruled out.^94,95

            On histopathological examination,
subcorneal or intraepidermal, variable sized pustules containing neutrophils
and occasionally few acantholytic cells, associated with spongiosis and
neutrophilic exocytosis are observed (Figure 4.12). In the superficial dermis,
there is a mild to moderate, perivascular to interstitial dermatitis.
Amastigotes may be occasionally identified in the dermis underneath the
pustules, but not within the pustules, by means of immunohistochemistry
(IHC).^94,95

4.6. Footpad and/or nasal
hyperkeratosis

            Footpad and/or nasal hyperkeratosis is characterized by greyish, thick,
and dry scales (Figure 4.13). These are strongly adherent to the underlying
epidermis and sometimes are accompanied by deep fissures, which can be painful,
especially when located on the paw pads.⁷⁰ This dermatological problem is often associated with other clinical
manifestations of CanL in moderately to severely affected dogs.⁷⁰  Recently, it has been suggested
that the combination of alopecia and nasal hyperkeratosis showed the greater
positive likelihood ratio to increase the pre-test probability of CanL.⁹⁶ Histopathologic findings seen in the hyperkeratotic footpads include
epidermal hyperplasia with hyperkeratosis, epidermal hypermelanosis, melanin
incontinence, perivascular to interstitial and, less commonly, nodular to
diffuse dermatitis.⁹⁷ The main inflammatory cells are macrophages and to a lesser extent
lymphocytes and plasma cells, whereas fewer neutrophils, eosinophils and mast
cells may be present.⁹⁷

4.7. Onychogryphosis

            Onychogryphosis is common in CanL, with a reported prevalence between
43.4% and 54.4%.⁷⁰ It is a tardive, chronic sign of CanL, and it is clinically characterized
by excessive growth and abnormal curvature of the nails (Figure 4.14). Rarely,
it is the only clinical sign, because in most dogs with CanL it is accompanied
by exfoliative and/or ulcerative dermatitis. Onychogryphosis, as well as
footpad and/or nasal hyperkeratosis, may represent a localized form of
exfoliative dermatitis.^34,98

            Histopathological findings are
non-specific. Onychogryphosis is characterized by lymphocytic exocytosis, mild
to severe lichenoid mononuclear dermatitis with or without hydropic
degeneration of basal keratinocytes, dermo-epidermal clefting, and pigmentary
incontinence.⁹⁸ Amastigotes cannot be found, at least in H&E stained preparations.⁹⁸

5. Diagnosis of canine
leishmaniosis

            The investigation of infection by L. infantum and/or for CanL is
performed for different indications and reasons, including suspected CanL in dogs with compatible
clinical signs, evaluation of blood donors and breeding stock, health check of
clinically healthy dogs, importation or exportation of dogs, monitoring dogs
during treatment, and epidemiological surveys.

            </span;>Several types of tests are available ranging from techniques to
visualize the parasites, to the detection of antibodies against Leishmania,
and to the molecular detection of parasite DNA.²³ These techniques have different sensitivities and specificities. Some
are more useful for particular purposes, such as the diagnosis of CanL in dogs
with compatible signs admitted for veterinary care, whereas others are more
valuable for the detection of subclinical infection in blood donors or
apparently healthy dogs that are undergoing a health check. The diagnostic
approach to a dog suspect of CanL should include, at minimum, complete blood
count, serum biochemical profile, urinalysis (including UPC) and one or more of
the following tests.^99,100

5.1. Microscopy and histopathology

            Leishmania amastigotes can be
demonstrated by microscopic examination of smears from the skin, lymph nodes,
spleen, bone marrow, joint fluid, abdominal fluid or other fluids, tissues, and
organs. The preparations should be stained with Romanowsky type stains, such as
Giemsa or Diff Quik^TM. Amastigotes are round to oval, 2.5 to 5 μm
long and 1.5 to 2 μm wide, and they possess a nucleus and a rod-shaped, darker
staining kinetoplast that is visible in the cytoplasm separately from the
nucleus (Figure 5.1). The
diagnostic sensitivity of microscopy depends on the parasitic load in the
target tissue, the quality of the preparations, the experience of the examiner
and the number of microscopic fields that are examined. In general, the
sensitivity is much higher in dogs with CanL compared to subclinically infected
dogs. For example, the sensitivity of lymph node cytology in dogs with CanL was
found to be 84% or 93% after the examination of 100 or 1,000 microscopic
fields, whereas the relevant figures for subclinically infected dogs were 13%
and 26%, respectively.¹⁰¹ Amastigotes may also be viewed in histopathological and/or IHC
examination of skin and other organs (see Diagnostic approach to the skin
lesions of dogs with leishmaniosis).⁹⁹

5.2. Serology

            Several serological methods for the detection of anti-Leishmania
antibodies are available. These include quantitative tests, such as the indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and direct agglutination test (DAT), as well as
qualitative commercial kits. The latter provide only a positive or negative
result, and when positive, they should be followed by a quantitative test,
which will provide a titer that is important for treatment monitoring
(successful treatment is typically associated with a decrease of antibody
levels over time, whereas unsuccessful treatment or relapses are associated
with increased antibody levels).¹⁰² All these tests employ whole parasites or recombinant antigens for the
detection of Leishmania-specific IgG. In general, good sensitivities and
specificities are gained with most serological assays for the diagnosis of
CanL, whereas subclinically infected dogs are often seronegative or have low
antibody levels.⁹⁹ In regions where multiple Leishmania species and/or Trypanosoma
spp. coexist, serological cross-reactions may occur.^103,104

5.3. Molecular tests (PCR)

            These tests allow the diagnosis of infection with Leishmania spp.
by the detection of parasite DNA. Many assays that target different sequences
of genomic or kinetoplast DNA (kDNA) have been developed, and, generally, those
targeting kDNA are the most sensitive. PCR can be performed on DNA extracted
from tissues, blood, other fluids such as cerebrospinal or synovial liquid, or
even from histopathologic specimens. The biological samples that are
characterized by the higher sensitivity are bone marrow, lymph node and spleen
aspirates, as well as conjunctival swabs, with the latter being the only
samples that are obtained non-invasively. PCR using blood and other body fluids
is considered less sensitive and dogs with CanL can be negative.¹⁰⁵ On the contrary, PCR of bone marrow and lymph nodes is typically
positive in dogs with CanL, and it can also be used for detection of Leishmania
in subclinically infected seronegative dogs.^99,100

Consensus statement:
The diagnosis
of CanL is based on the compatible clinical signs and/or clinicopathologic
abnormalities, the exclusion of as many differentials as possible, the
demonstration of infection and the increased concentration of anti-Leishmania
IgG in serum (quantitative serology). Both PCR and
serology, in combination, can detect subclinical infection in blood donors,
breeding dogs, dogs before importation to non-endemic countries, and in epidemiological
studies, whereas serology is probably adequate for health checks.

5.4. Staging of canine leishmaniosis

            Two
main non-validated systems have been proposed for staging of CanL.^99,106,107 The clinical
staging system for CanL provided by the LeishVet group can be found at https://www.leishvet.org/fact-sheet/clinical-staging/. It divides the disease into four stages based on clinical signs,
clinicopathologic abnormalities and level of anti-Leishmania antibodies.
This system is helpful for decisions on the most suitable treatment for each
dog, and for consideration of a prognosis. The clinical stage may change if the
dog improves or deteriorates.^99,100

6. Diagnostic approach to the skin lesions
of dogs with leishmaniosis

            A
dog with CanL may present no macroscopic skin lesions, may present skin lesions
that are directly or indirectly caused by CanL, or it may present skin lesions
due to coincidental diseases.⁷⁰ The precise diagnosis of the cause of skin lesions in a dog with CanL
is of major importance for the overall management and prognosis. As an example,
treatment and prognosis of a dog with CanL and scaling will be vastly different
if the scaling is due to CanL-associated exfoliative dermatitis compared to
scaling due to concurrent epitheliotropic T cell lymphoma. Unfortunately, the
polymorphism of CanL-associated skin lesions results in an extensive list of
differentials (Table 4.1) that becomes even longer when different types of skin
lesions are present in the same dog (e.g., when a dog with CanL shows
exfoliative dermatitis, ulcerative dermatitis, and footpad hyperkeratosis).⁷⁰

            The
laboratory examinations that can be used to diagnose CanL-associated skin
lesions include cytology, histopathology, IHC, direct immunofluorescence (DIF),
and PCR. The necessity to undertake some or all these examinations depends on
the certainty of CanL diagnosis, the macroscopic appearance of skin lesions,
the clinical experience of the veterinary surgeon, and the geographical area.
For example, after a definitive diagnosis of CanL, few to no additional
examinations may be needed for a dog that lives in an endemic area and presents
the typical lesions of CanL-associated exfoliative dermatitis. On the contrary,
if the diagnosis of CanL is not certain (e.g., it is based only on positive
qualitative serology), the clinical presentation is not typical and/or the dog
lives in a non-endemic area, an extensive diagnostic investigation should
follow to confirm that the skin lesions are due to CanL and not to another
concurrent disease.

            Cutaneous
cytology can demonstrate the inflammatory component of CanL skin lesions
(typically macrophagic or purulent-macrophagic), confirm the presence of
intracellular or extracellular Leishmania amastigotes (Figure 6.1), and
help to exclude or confirm some differentials, such as superficial and deep
bacterial infections, deep fungal infections, pemphigus foliaceus, neoplastic,
and non-neoplastic tumors. Samples can be obtained from areas with exfoliative
dermatitis after gentle lifting the scales and crusts, from the border of
ulcers, from papules or nodules after fine needle puncture or fine needle aspiration,
and from skin biopsy samples (imprint smears).^108-110 When microscopy is performed by experienced examiners, the detection of
amastigotes has a specificity of 100%,^111-113 but the sensitivity depends not only on the examiner, but also on the
time devoted to review the slide (i.e., number of fields examined), and the
type of macroscopic lesions.¹⁰¹ In CanL, the diagnostic sensitivity of cutaneous cytology varies from
62% to 100%,^113,114 it may be
higher in exfoliative compared to ulcerative dermatitis¹⁰⁸ (in the latter it is up to 36%),¹¹⁵ in the papular form varies from 33% to 62%,^87,88 and in the
nodular form it is typically very high.¹⁰⁸

            Histopathological
examination of skin biopsies (Figure 6.2) will show CanL-associated lesions in
the epidermis, dermis, and panniculus, characterize the type and distribution
of the inflammatory infiltrate, that vary depending on the macroscopic lesions
(reviewed in⁷⁰), and help to exclude or confirm many differentials (Table 4.1).
However, it may be difficult to visualize the organisms, especially when their
number is low, and it is almost impossible to definitively identify them as Leishmania
amastigotes because their characteristic features (cell membrane, nucleus,
kinetoplast) are not readily visible.^116,117 Using Giemsa
instead of H&E stain may help in the identification of the parasites.¹¹⁸ In general, presence of presumed amastigotes is reported in 7-75% of
dogs with various skin lesions due to CanL,^{89,97,117,119-123} and in 9% of
dogs with papular dermatitis.⁸⁹

            Immunohistochemistry
(Figure 6.3) is a useful adjunct to histopathology because it can detect low
number of organisms,^122,124 prove their
identity as Leishmania amastigotes, and, thanks to the counterstain,
show their localization within inflammatory foci. Although the specificity of
meticulously standardized IHC protocols is high, there are still some doubtful
cases where dye precipitates cannot be easily differentiated from low numbers
of amastigotes.¹²⁵ Sensitivity depends on the parasitic density,¹²⁶ may be higher than
that of cytology,¹¹⁷ varies from 18% to 100% in dogs with CanL and various skin lesions,^{89,117,118,122,123,127} and has been
reported to be 31% in exfoliative dermatitis⁷⁶ and 82-100% in papular dermatitis.^87,89

            Direct
immunofluorescence (Figure 6.4) has been proposed as an alternative to IHC. Due
to the lack of background staining it permits more accurate measurement of the
parasitic density in the skin,⁹⁷ but gives no information about the location of the parasites in
relation to the areas of inflammation. Although in the single published study
on this topic the sensitivity of DIF was 100%,⁹⁷ it was not compared with the sensitivity of IHC. Τhe specificity of DIF is unknown, and currently it is not commercially
available.

            Skin
PCR (conventional PCR, nested PCR, qPCR) is probably the most sensitive test
for the detection,^{88,89,113,117,125,127-129} and the most
accurate test for the quantification (qPCR) of Leishmania DNA,^130-133 that presumably,^134-136 but not necessarily,^137,138 corresponds
to the presence of live amastigotes. However, it gives no information on the
location of amastigotes in the skin, and it may be positive in transiently
infected dogs (e.g., if the biopsy sample was obtained from an area recently
bitten by an infected vector), in subclinically infected dogs, and in dogs with
parasitemia (due to the inevitable presence of blood in the biopsy material).^139,140
Alternatively, skin scrapings can be used instead of biopsy samples, but in
this case the sensitivity of PCR may be significantly lower.¹⁴¹

            In
conclusion, minimally invasive, low-cost diagnostic tests, such as cytology and
parasitological examinations, should be performed in every dog with CanL and
skin lesions; they may strengthen (or weaken) the possibility that the skin
lesions are due to CanL (e.g., cytology) and help to confirm or exclude other
diseases that may coexist (e.g., demodicosis, pemphigus foliaceus, pyoderma).
More invasive and costly examinations (skin biopsy for histopathology, IHC or
qPCR) should be considered when the diagnosis of CanL is not definitive (i.e.,
it is based only on positive qualitative serology), the skin lesions are the
only findings (i.e., dogs without systemic signs and clinicopathological
abnormalities) especially in non-endemic areas, and the macroscopic appearance
of skin lesions is not typical of CanL and/or is compatible with concurrent
skin diseases.

6.1. Case example 1

            Penny
is a 5-year-old, spayed-female, Jack Russell terrier, that about 5 months ago presented
with areas of partial alopecia, extending progressively from the head to the
dorsum, that were initially accompanied by moderate pruritus. Glucocorticoids
and oclacitinib improved pruritus but not alopecia. The dog is otherwise
healthy and a rapid immunochromatographic test for CanL was negative. Penny
lives indoors and outdoors in the garden, and regularly receives imidacloprid
and permethrin spot on.

            General physical
examination showed no abnormalities, and on dermatologic examination areas of
partial alopecia were observed on the head, dorsal trunk, and outer surface of
the hind limbs (Figures 6.5-6.6). The cutaneous problem was defined as
multifocal partial alopecia and the diagnostic hypotheses included
demodicosis, idiopathic sebaceous adenitis, CanL, hypothyroidism, and less
likely, immune mediated-autoimmune folliculitis.

            No
parasites were observed on microscopic examination of skin scrapings and
plucked hairs. Complete blood count, biochemistry profile with serum protein
electrophoresis, urinalysis, quantitative serological test (ELISA) for CanL,
and measurement of serum total T4 revealed mild hyperglobulinemia [3.6 g/dL; reference range (RR): 2.7-3.5 g/dl] and a low-positive ELISA (antibody level 14.8%;
positivity cut-off >11%, with values between 11 and 30% considered as
low-positive).

            A
bone marrow qPCR was performed, and multiple skin biopsies were taken to
confirm or rule out sebaceous adenitis. Bone marrow qPCR was negative, and the
main cutaneous histopathologic lesions were periadnexal, histiocytic and
lymphoplasmacytic infiltrates with less neutrophils, associated with
disappearance of sebaceous glands, and mild-to-moderate basketweave and
lamellar orthokeratotic hyperkeratosis with follicular keratosis. The lesions
were compatible with idiopathic sebaceous adenitis. However, since the dog
lived in an area endemic for leishmaniosis and CanL can cause sebaceous
adenitis, and because of the presence of rare small granulomatous foci in the
panniculus, skin PCR for Leishmania was performed and was negative.

            It
was concluded that Penny is a seropositive dog with idiopathic sebaceous
adenitis; however, it was considered essential to periodically monitor the
patient over time by serologic testing, especially if ciclosporin was to be
administered for the management of idiopathic sebaceous adenitis.

            Seropositive
but PCR-negative dogs, without clinical signs or clinicopathologic changes, that
reside in or have visited an area where sand fly vectors are present, must be
monitored over time. Recently, it has been reported that nearly one fourth of
clinically healthy seropositive dogs living in an endemic area will become
seronegative by the end of the next non-transmission season.¹⁴² Probably these dogs represent a heterogeneous group, including infected
dogs with a low parasite load that is non-detectable by PCR even in tissues
where their numbers are normally high, such as bone marrow and skin, and dogs
with a transient infection. Also, seropositivity in dogs in which infection
cannot be demonstrated has been attributed to nonspecific false-positive reactions,^{104,140,143,144} and to
cross-reactions due to infection by other trypanosomatids, including pathogenic
(e.g. L. braziliensis) and non-pathogenic (e.g. L. tarentolae) Leishmania
species.^145,146 On the
contrary, evidence for cross-reactivity with other pathogens (e.g. Ehrlichia canis, Toxoplasma gondii, Neospora
caninum and Babesia canis) is
weak and speculative.

6.2. Case example 2

            Nina
is an 8-month-old, spayed-female, Labrador retriever that for about 2 months shows
papules on the inner side of both ear pinnae. These lesions have grown larger
in the last few days. Nina was initially treated with a cream containing
gentamicin and dexamethasone without improvement. It is otherwise healthy,
lives indoors and outdoors in a house, regularly receives a spot-on product
containing fipronil and is wearing a deltamethrin-impregnated collar.

            General
physical examination showed no abnormalities, and dermatologic examination
revealed multiple, whitish, 3-6 mm papules on each ear pinna (Figure 6.7). The
cutaneous problem was defined as persistent papular dermatitis on both inner
ear pinnae and the diagnostic hypotheses included CanL, insect bites, and
canine leproid granuloma syndrome.

            Cytologic
examination of samples obtained by fine-needle puncture revealed scarce
lymphocytes and macrophages containing few Leishmania amastigotes in
their cytoplasm (Figure 6.8). Complete blood count, biochemistry profile with
serum protein electrophoresis, and urinalysis did not show alterations, and
quantitative serological test (ELISA) for leishmaniosis was negative (13.2
ELISA units; positivity cut-off >35).

            If
amastigotes had not been detected on cytological examination, one of the
following could have been performed: a) Leishmania qPCR from material
collected through impression, fine needle puncture or scraping from the surface
of the papules, or b) skin biopsy for histopathology followed, if necessary, by
IHC or qPCR.

            Nina
was affected by a mild form of CanL, namely papular dermatitis. Information
regarding the treatment and outcome of this form is scarce. However, the
prognosis is good, even without treatment. Nonetheless, physical examination
and humoral immune response monitoring is needed to detect early disease
worsening.

Nina was left
untreated. Ten days after diagnosis papules evolved to crusted papules with an
umbilicated appearance (Figure 6.9). Twenty-five days after diagnosis the
lesions were regressing and were completely resolved at 37 days (Figure 6.10).
One month after diagnosis the results of hematology, serum biochemistry and
urinalysis were within RR, and ELISA was still negative and remained so one
year after diagnosis and on annual rechecks in the following 4.5 years.

6.3. Case example 3

            DJ
is an 8-year-old, neutered-male, American Staffordshire terrier with
generalized skin lesions since approximately one year. DJ was initially
diagnosed with atopic dermatitis, with episodes of pruritus, especially during
the summer, and was receiving allergen-specific immunotherapy. A recent
worsening was witnessed, and the dog was treated with oral cephalexin and oclacitinib
without improvement. Fifteen days before consultation, complete blood count and
biochemistry profile with serum protein electrophoresis were performed and
revealed hypoalbuminemia (2.4 g/dL; RR: 2.87-4.76 g/dl), hyperbetaglobulinemia
(2.4 g/dL; RR: 0.72-1.80 g/dl), hypergammaglobulinemia (2.4 g/dL; RR: 0.28-1.57
g/dl) and a mild increase in aspartate aminotransferase activity (130 IU/L; RR:
16-89 UI/L). Quantitative serology for CanL (ELISA) was very high positive
(R=1.82; R >1.8 = very high positive). The patient lives in an apartment
without any other pets and regularly receives oral afoxolaner.

            General
physical examination showed moderate popliteal lymphadenomegaly, and
dermatologic examination revealed skin lesions mainly on the head, ear pinnae,
and limbs (Figure 6.11). Alopecia with fine whitish scales was observed on both
pinnae (Figure 6.12), periocular region (Figure 6.13), elbows and tarsal
regions (Figure 6.14). Ulcers were observed on the inferior lip and tongue
margins (Figure 6.15). The cutaneous problems were defined as exfoliative
(scaling) dermatitis and mucocutaneous ulcerative dermatitis and the diagnostic
hypotheses included CanL, less likely a combination of CanL with atopic
dermatitis, demodicosis, exfoliative cutaneous lupus erythematous, idiopathic
sebaceous adenitis and hypothyroidism; for all these differentials, the
possibility of secondary bacterial infection was also considered.

            Microscopic
examination of plucked hairs was negative for mites. Cytological examination of
impression smears from the skin underneath scales revealed neutrophils with
intracellular cocci and a few macrophages. Urinalysis revealed urine specific
gravity of 1,040 and proteinuria (UPC: 2.64; RR <0.2).

            It
was concluded that DJ was affected by a severe clinical form of CanL associated
with secondary bacterial infection.

            DJ
was treated with meglumine antimoniate, at 100 mg/kg (divided every 12 hours),
subcutaneously (SC), and with allopurinol at 10 mg/kg, twice daily, and bathed
twice a week with chlorhexidine shampoo. One month later, dermatological
examination revealed scarring alopecia on the lips and periocular region and
mild interdigital erythema. Biochemistry profile with serum protein
electrophoresis revealed mild hypoalbuminemia (2.67 g/dL; RR: 2.93-4.12 g/dl),
hypergammaglobulinemia (1.53 g/dL; RR: 0.24-0.86 g/dl) and improved proteinuria
(UPC: 1.69).

7. Systematic review on treatment
and prevention of canine leishmaniosis

            A systematic review of all,
positive- or placebo-controlled, randomized controlled trials (RCTs) and of
up-to-date (i.e., published between 2018 and 2022) systematic reviews or
meta-analyses on the treatment and prevention of CanL was done. To this aim, RCTs,
systematic reviews and meta-analyses on the efficacy of one or more therapeutic
and/or preventive interventions in dogs with natural CanL, published in peer-reviewed
journals in any language (articles published in non-English language should
have had an English abstract to be considered), were evaluated.

To be eligible, RCTs on the treatment of CanL had to include naturally
infected dogs presenting clinical signs and/or common and clinically important
laboratory abnormalities (i.e., anemia, hypoalbuminemia, hyperglobulinemia,
proteinuria) compatible with the disease, and the diagnosis of CanL had to be
confirmed by demonstration of the infection (e.g., by molecular tests,
cytology, histopathology and/or immunohistochemistry) and/or by positive
serology. RCTs including both dogs with CanL and subclinically infected dogs were
eligible if the results for the former could be clearly differentiated from the
results for the latter dogs.

To be eligible, RCTs on the prevention of CanL had to include dogs with
no evidence of infection and/or subclinically infected dogs not presenting
clinical signs and/or laboratory abnormalities compatible with CanL. RCTs
including both subclinically infected dogs and dogs with CanL were eligible if
the results for the former could be clearly differentiated from the results for
the latter dogs.

            Relevant articles were searched in Medline
(via PubMed), Thomson Reuter’s Web of Science) and CAB Abstract (via EBSCO host)
on January 11^th 2023 using the following search string: “(dog OR
dogs OR canine) AND (leishman*) AND (treatment OR
therapy OR trial OR prevent* OR antimon* OR meglumin* OR stibogluconate* OR allopurinol* OR miltefosin* OR aminosidin* OR
paromomycin* OR amphotericin* OR pentamidin* OR ketoconazol* OR itraconazol* OR fluconazol* OR metronidazol* OR
azole OR spiramycin* OR terbinafin*
OR diminazen* OR phosphocholin* OR furazolidon* OR sitamaquin* OR fluoroquinolon* OR enrofloxacin* OR marbofloxacin* OR
trifluralin* OR bisabolol OR levamisol*
OR mycobacterium OR interferon* OR interleukin* OR impromun*
OR domperidon* OR glucocorticoid* OR corticosteroid*
OR predniso* OR antimicrobial peptide OR cecropin OR
melittin OR insecticid* OR repellent OR mesh net OR
pyrethroid* OR deltamethrin* OR permethrin* OR flumethrin* OR cyhalothrin* OR
oil OR citronella OR deet OR fenthion* OR diazinon* OR pyriprol*
OR fipronil* OR imidacloprid* OR metaflumizon* OR
amitraz OR spinosad* OR isoxazolin*
OR afoxolaner OR fluralaner OR lotilaner OR sarolaner
OR pheromon* OR vaccin* OR canileish OR letifend OR leishvaccine OR leishmune OR leish-tec OR leishtec)”. Since the previous systematic review included all relevant
articles that were published between 1980 and 2004,¹⁴⁷ our search was limited
to articles published before 1980 and after 2004 and included publications
available as early-view articles published electronically ahead of printing, on
2022. The search results were tabulated and
cross-checked by two authors. The titles, abstracts and, when necessary, the
full texts of these articles were scrutinized independently by two authors to
identify those fulfilling the above eligibility criteria.

For each of the therapeutic interventions, data were initially extracted
and tabulated by one author and then they were cross-checked by another author.
Data of interest included the following: i) number of dogs; ii) clinical
status/severity of CanL (using any classification/scoring system); iii) method
of diagnosis of CanL; iv) dosage regimen (dose, route of administration, frequency
of administration, duration); v) the percentage (%) of dogs that achieved
clinical cure (defined as absence of clinical signs) and the time to achieve
clinical cure; vi) the % of dogs that achieved clinical improvement without
cure, the time to achieve clinical improvement and the degree of clinical
improvement (using any clinical scoring system); vii) the % of dogs that
achieved either clinical cure or improvement and the time to achieve clinical
cure or improvement; viii) the % of dogs that dropped out of the study, in RCTs
without intention-to-treat (ITT) analysis of the results; ix) the % of dogs
that died or were euthanized; x) the % of dogs that relapsed after treatment
discontinuation and the time to relapse; xi) the % of dogs with normalization
of clinically important laboratory abnormalities [i.e., anemia,
thrombocytopenia, increased total proteins, decreased albumins, increased
globulins, decreased albumin/globulin ratio, protein electrophoresis abnormalities,
increased blood urea nitrogen (BUN), increased creatinine, increased SDMA,
increased inorganic phosphorus, increased CRP or other acute phase proteins,
proteinuria] and the time for normalization; xii) the % of dogs with
improvement without normalization of the above laboratory abnormalities and the
time for improvement; xiii) the % of dogs with either normalization or
improvement of the above laboratory abnormalities and the time for
normalization or improvement; xiv) the % change of packed cell volume (PCV) and/or
of hematocrit and/or of hemoglobin concentration (Hb), of platelet count, total
proteins, albumins, globulins, albumin/globulin ratio, protein fractions (based
on protein electrophoresis), BUN, creatinine, SDMA, inorganic phosphorus, CRP
or other acute phase proteins, and UPC; xv) the % of dogs with reappearance of the
above laboratory abnormalities after treatment discontinuation and the time for
reappearance; xvi) the reduction of parasitic load, the examined organ(s) or
tissue(s), the method and the time of evaluation; xvii) the % of dogs with
absence of parasites, the examined organ(s) or tissue(s), the method and the
time of evaluation; xviii) the increase of parasitic load after treatment
discontinuation, the examined organ(s) or tissue(s), the method and the time of
evaluation; xix) the changes in the number of immune cells and/or of  lymphocyte subpopulations and the induction
of cell-mediated immunity, including the method [e.g., leishmanin skin test,
lymphocyte proliferation assays, cytokine production by peripheral blood
mononuclear cells (PBMCs), leishmanicidal activity of macrophages, etc.] and
the time of evaluation; xx) the reduction of antibody titer or optical density
(OD), the method and the time of evaluation; xxi) the % of seropositive dogs
that became seronegative, the method and the time of evaluation; xxii) the increase
of antibody titer or OD after treatment discontinuation, the method and the time
of evaluation; xxiii) the reduction of parasite transmission to sand flies, the
method and the time of evaluation; and xxiv) the adverse effects of the
treatment, their frequency and their severity.

For each of the preventive interventions, data were initially extracted
and tabulated by one author and then they were cross-checked by another author.
Data of interest included the following: i) number of dogs; ii) clinical status
of dogs: subclinical infection and/or seropositivity or no evidence of
infection, including the method(s) of evaluation; iii) dosage regimen (dose, route
of administration, frequency of administration) if applicable; iv) the % of
subclinically infected dogs that did not develop CanL and/or did not develop
clinical signs of CanL and/or did not develop laboratory abnormalities of CanL;
v) the % of seropositive dogs and the % of seronegative dogs that did not
develop CanL and/or did not develop clinical signs of CanL and/or did not
develop laboratory abnormalities of CanL; vi) the % of dogs with no evidence of
infection that did not develop CanL and/or did not develop clinical signs of
CanL and/or did not develop laboratory abnormalities of CanL; vii) the severity
of clinical signs and laboratory abnormalities of CanL (using any
classification/scoring system) for those dogs that developed the disease; viii)
the immunological changes, such as changes in the number of immune cells and/or
of  lymphocyte subpopulations and the
induction of cell-mediated immunity, including the method of evaluation (e.g.,
leishmanin skin test, lymphocyte proliferation assays, cytokine production by
PBMCs, leishmanicidal activity of macrophages, etc.); ix) the reduction of
parasitic load of subclinically infected dogs, the examined organ(s) or
tissue(s), the method and the time of evaluation; x) the % of subclinically
infected dogs and the % of seronegative dogs with absence of parasites, the
examined organ(s) or tissue(s), the method and the time of evaluation; xi) the
% of dogs with absence of parasites both before and after the intervention, the
examined organ(s) or tissue(s), the method and the times of evaluation; xii) the
reduction of antibody titer or OD of seropositive dogs, the method and the time
of evaluation; xiii) the % of seropositive dogs that became seronegative, the
method and the time of evaluation; xiv) the % of seronegative dogs that
remained seronegative, the method and the times of evaluation; xv) the
induction of vaccine-induced antibodies; xvi) the reduction of parasite
transmission to sand flies, the method and the time of evaluation; and xvii)
the adverse effects of the treatment, their frequency and their severity.

7.1. Evaluation
of quality of randomized controlled trials

            The quality of each RCT was
evaluated according to Olivry and Bizikova 2013¹⁴⁸ with some modifications.

            For each RCT on therapeutic
interventions, the following parameters were assessed as “adequate”, “unclear”
or “inadequate”: i) degree of certainty that all dogs present CanL and no
concurrent diseases (Table 7.1); ii) method of generation of randomization
sequences; iii) method of concealment of allocation to treatment groups; and
iv) inclusion of cases lost to follow-up in ITT analyses.

            For each RCT on preventive
interventions, the following parameters were assessed as “adequate”, “unclear”
or “inadequate”: i) degree of certainty that all dogs did not present CanL
(Table 7.2); ii) method of generation of randomization sequences; iii) method
of concealment of allocation to treatment groups; and iv) inclusion of cases
lost to follow-up in ITT analyses.

Each RCT on therapeutic or preventive interventions was rated as: i) high
quality when all four parameters were assessed as adequate; ii) intermediate
quality when at least one parameter was assessed as adequate and the remaining parameters
as unclear and/or inadequate; and iii) low quality when all four parameters were
assessed as unclear and/or inadequate.

7.2. Evaluation
of level of evidence and consistency among studies

            The level of evidence (LoE) was evaluated,
according to Ebell et al., 2004,¹⁴⁹ as simplified by Bond et al., 2020,¹⁵⁰ as: i) good quality, patient-oriented when
based on high quality RCTs or meta-analysis of consistent RCTs; ii) limited
quality, patient-oriented when based on lower quality clinical trials, cohort
studies or case-control studies; and iii) other evidence when based on
consensus, usual practice, disease-oriented evidence, or case series.

            For interventions that have been
tested in more than one RCT, the consistency of the results across studies was
evaluated, according to Ebell et al., 2004,¹⁴⁹ as: i) consistent when most studies found
similar or at least coherent (i.e., differences were explainable) results OR when
results are based on high-quality and up-to-date systematic reviews or
meta-analyses; and ii) inconsistent when there is considerable variation among
study results and lack of coherence OR when high-quality and up-to-date
systematic reviews or meta-analyses do not find consistent evidence.

7.3. Strength of
recommendation

Based on the quality of RCTs, LoE
and consistency among studies, the strength of recommendation (SORT) was
characterized^149,150 as i) strong when based on consistent and good
quality patient-oriented evidence; ii) moderate when based on inconsistent or
limited quality patient-oriented evidence; or iii) weak when based on consensus,
usual practice, disease-oriented evidence, or case series.

7.4. Eligible
randomized controlled trials

            The previous systematic review
included 60 references,¹⁴⁷ and search of the three electronic databases
yielded 2,700 additional articles, resulting in a total of 2,760 articles. At
initial screening 2,697 were excluded, and 63 articles were considered further.
Of these 63 articles, six were excluded for one of the following reasons: systematic
review evaluating only one RCT that has been included as separate publication
(n=1), RCT on the treatment of CanL not including dogs with clinical signs
and/or laboratory abnormalities due to CanL (n=1), RCT on the treatment of CanL
including both dogs with CanL and subclinically infected dogs because the
results for the former could be clearly differentiated from the results for the
latter dogs (n=1), and RCTs on the treatment or prevention of CanL not
reporting data of interest for this systematic review (n=3). Therefore, 57 articles
(55 RCTs and two meta-analyses) were evaluated (Figure 7.1).

8. Treatment of
canine leishmaniosis

8.1. Indications for euthanasia (instead of
treatment)

            Euthanasia of dogs with CanL (and,
even more, of subclinically infected seropositive dogs) has been advocated, and
is still enforced by the legislation of some countries, as an effective measure
to decrease the incidence of human VL due to L. infantum. The rationale
behind this approach is that in endemic areas, dogs are the major peridomestic
reservoir of the parasite and the primary source of its transmission to
vectors. There is some evidence in favor of the efficacy of this strategy in a
few countries, like China, after periods of massive eradication of all dogs (irrespectively
of their infectious and serology status) combined with extensive use of
environmental insecticides.¹⁵¹ However, this approach could possibly block
the transmission cycle of L. infantum only if seropositive dogs were the
sole reservoir capable to transmit the parasite, if all seropositive dogs could
be identified and euthanized, and if euthanized dogs were not replaced by new
ones that may also become reservoirs. Nowadays, it is clear that none of these
conditions holds true: other domestic animals (e.g., cats)^152-155 and wildlife can infect sand fly vectors and
can sustain or greatly amplify parasite transmission (e.g., hares and rabbits
in Madrid),^17,18 some subclinically infected seronegative dogs
may also transmit L. infantum,^156,157 and euthanasia of owned dogs is commonly
declined by their owners.¹⁵⁷ Moreover, identification of seropositive stray
dogs, which are numerous in many endemic areas, is impractical and
labor-intensive,¹⁵⁶ massive euthanasia of dogs is, at minimum,
ethically questionable and opposed to the role of these animals in modern
society,¹⁵⁸ and the euthanized dogs are frequently
replaced by new ones.^159,160 All the above explain the results of
epidemiological models showing that euthanasia is the least effective measure
for the control of human VL due to L. infantum, the reoccurrence of the
disease in China, and the limited efficacy of the euthanasia- plus
environmental insecticide-based VL control program in Brazil.^158,161-165

            A second argument in favor of
euthanasia is that the alternative option, namely the treatment of dogs with
CanL, will induce drug-resistant strains of L. infantum that may result
in cases of human VL unresponsive to the same and to cross-resistant drugs.
However, in addition to the great reduction of infectivity to sand flies during
treatment of CanL,¹⁶⁶ the avoidance of first-line drugs for
treatment of human VL in the same area along with the systematic use of insect
repellents and administration of isoxazolines during and after treatment (see
prevention), makes the justification of this argument very difficult.

            Finally, it has been proposed that
euthanasia of dogs with CanL (or of all infected dogs) living in non-endemic
areas may prevent the establishment of the infection.¹⁶⁷ If such an area is close to an endemic one,
the environmental conditions are favorable for sand fly vectors, and there is
an adequate number of susceptible dogs, spread of the infection seems
unavoidable.^7-9 On the other hand, in the absence of sand fly
vectors, infection can be eradicated if infected dogs are removed from the
breeding stock and are not used as blood donors.^25,26

            Despite the limited efficacy of
euthanasia in terms of public health and of geographical containment of the
infection, there are circumstances where it is indicated or may be considered
on ethical grounds, such as: a) inability to offer optimal treatment and to
systematically apply transmission blocking measures (e.g., owner’s refusal due
to financial or other reasons, stray dogs) to dogs with CanL at a stage where
self-cure is unlikely; b) poor prognosis, usually due to advanced chronic
kidney disease (CKD) and less commonly due to liver failure; c) poor quality of
life despite treatment.

Conclusion: Euthanasia of dogs with CanL is not recommended
as a tool to decrease the incidence of human VL due to L. infantum, to
avoid induction of drug-resistant parasites and to block the expansion of
endemic areas (SORT: moderate). However, euthanasia of individual dogs with
CanL can be considered if proper treatment cannot be administered and prognosis
or their quality of life is poor (SORT: weak).

8.2. Indications and aims of treatment

            With the exceptions listed in the
previous section, all dogs with CanL should be treated with drugs having direct
anti-Leishmania activity, with immunostimulants or with their
combination. Routine treatment of subclinically infected dogs, especially those
with high or increasing antibody titers, has also been proposed because of a
perceived high risk to develop CanL.¹⁶⁸ This practice is discouraged since the
widespread use of drugs with direct anti-Leishmania activity promotes
drug-resistance,¹⁶⁹ and the majority of subclinically infected
seropositive dogs will not develop CanL, at least during the next 8-12 months.^142,170 However, administration of immunomodulators,
like domperidone or dietary nucleotides plus active hexose correlated compound
(AHCC) that cannot induce drug-resistance and are generally safe, should be
considered,^170,171 in addition to the regular clinical and
clinicopathological monitoring (more frequently than every 4 months, especially
for dogs with moderate-to-high antibody titers), and use of insect repellents
with proven efficacy against sand flies biting.^107,170,172

            Treatment of CanL should lead to
clinical cure (resolution of clinical signs and important clinicopathologic
abnormalities, such as anemia and proteinuria), halt the progression and, if
possible, reverse organ damage, reduce parasitic load, avoid the induction of
drug-resistant strains of L. infantum, minimize the infectivity of dogs
to sand flies, promote a strong and long-lasting Leishmania-specific
cell-mediated immunity that will prevent disease recurrence, and be safe.¹⁷³ Complete elimination of the parasite
(parasitological cure) is rarely, if ever, achieved, is meaningless in endemic
areas because exposure to new sand fly bites cannot be completely avoided, and
may not be prudent, since a small number of viable parasites may contribute to
immunological memory.¹⁷⁴

            To this aim, the ideal drug (or drug
combination) for the treatment of CanL should be highly effective and safe
(based on the results of RCTs in dogs with natural CanL), administered orally
(to permit outpatient treatment and to avoid injection site adverse effects),
reasonably priced, registered for the treatment of CanL, and not be a
first-line drug for the treatment of human VL in the same area. Unfortunately,
a drug or a drug combination with all these attributes does not exist.

Conclusion: Drugs with direct anti-Leishmania
activity and/or immunostimulants should be used for the treatment of dogs with
CanL. Administration of drugs with direct anti-Leishmania activity
should be avoided in subclinically infected dogs (SORT: weak). The aim of the
treatment is not the parasitological cure, but the induction of Leishmania-specific
cell-mediated immunity (SORT: weak).

8.3. Drugs with direct anti-Leishmania activity

            The drugs with direct anti-Leishmania
activity that are used more commonly for the treatment of CanL include
pentavalent antimonials, particularly meglumine antimoniate, miltefosine, and
allopurinol.

8.3.1. Meglumine antimoniate

            Despite using pentavalent
antimonials for more than 70 years, the exact mechanism of their action remains
unclear. After entering cells, including host macrophages and Leishmania
amastigotes, pentavalent antimony (Sb^v) is reduced to the active
trivalent form that causes amastigote apoptosis, probably through inhibition of
ATP and GTP synthesis, change of the structure and function of glucosomes with
ensuing disturbance of glucose and fatty acid metabolism, inhibition of the citric
acid cycle, and inhibition of trypanothione reductase with subsequent thiol
loss.^175-177 Furthermore, antimonials may have additional
indirect anti-Leishmania effects, including increased macrophage and
neutrophil phagocytic and leishmanicidal activity.¹⁷⁸

            After SC administration in dogs,
meglumine antimoniate is completely absorbed, reaches maximum plasma
concentrations within 2-4 h, and is quickly eliminated with the urine, having a
serum half-life of approximately 2h.¹⁷⁹ However, antimony accumulates in macrophages
where it remains for at least 3 days,¹⁸⁰ and this permits administration once per day.
The recommended dose of meglumine antimoniate is 100 mg/kg (corresponding to
28.3 mg antimony/kg) SC, once daily or divided in two daily doses, for 28 days
(4 weeks).^{147,179,181-184} A dose decrement may be indicated in dogs with
CKD due to the anticipated reduced excretion of the drug.¹⁸⁵ 

            The efficacy and
safety of meglumine antimoniate in dogs with CanL have been evaluated in eight
RCTs (Table S1).^{181,184,186-191} In these studies, two routes of drug administration [SC
and intravenous(IV)] were compared;¹⁸⁴ meglumine antimoniate
was compared to miltefosine,¹⁸¹ aminosidine,¹⁸⁶ MTC-305 (an O-alkyl-hydroxamate
derivative),¹⁸⁷ (-)-a-bisabolol,¹⁸⁸ a vaccine containing LiF2 (a purified fraction
of L. infantum promastigotes),¹⁹⁰ two vaccines containing the adjuvant MPL-SE^®
plus recombinant antigens (Leish-110f^®189 or Leish-111f^®),¹⁹¹ and to placebo;^189,191 and
meglumine antimoniate monotherapy was compared to its’ combination with aminosidine,¹⁸⁶ MTC-305,¹⁸⁷ LiF2,¹⁹⁰ Leish-110f^® with MPL-SE^®,¹⁸⁹ Leish111f^® with MPL-SE^®,¹⁹¹ and MPL-SE^®.¹⁸⁹ In six of these studies the daily dose of the
drug was 100-106 mg/kg, the duration of treatment ranged from 20 to 28 days, in
two of them a second treatment “cycle” was administered one month after the end
of the first one,^187,188 and
in another study the second “cycle” was administered if there was not a
complete remission 3 weeks after the end of the first one.¹⁸⁴ In the remaining two RCTs, meglumine
antimoniate was administered at 20 mg/kg, once daily for 30 days,¹⁹¹ or at 300 mg/kg every second day for a total
of 20 administrations (i.e., 40 days).¹⁹⁰ The number of dogs with CanL treated with
meglumine antimoniate varied from six^187-189 to 57,¹⁸¹ the confirmation of CanL diagnosis was based
on serology (6/8)^{181,184,186-188,191}
and/or on the demonstration of parasite by microscopy and/or culture (6/8)^{181,184,186,189-191}
and/or on the demonstration of parasitic DNA with molecular methods (3/8),^181,187,188 the
severity of CanL is reported only in one study that included dogs at LeishVet
stages I, II, and III,¹⁸⁷ whereas in two other studies serum creatinine
concentration within reference range was an inclusion criterion.^181,184 The
quality of these studies is intermediate^{181,186-188,190,191} or
low.^184,189

            According to the results of these
studies, the efficacy varies, depending mainly on the time of evaluation: when
assessed at the end of treatment period, clinical cure or improvement was
witnessed in 81-100% of dogs;^184,186 when assessed 2 weeks after the end of treatment, total clinical score
was significantly (by 63.4%) lower compared to pre-treatment score;¹⁸¹ when assessed 4-5 months after the end of
treatment, clinical cure or improvement were recorded in 33.3-100% of dogs,^187-191 but the total clinical score was numerically
higher than before treatment,^187,188 and 0-33.3% of dogs had died of CanL;^187-191 and when assessed 3 years after the end of
treatment, clinical cure or improvement were recorded in 63.6% but 26.7% of dogs
had died of CanL.¹⁹¹ The difference between early and late efficacy
occurred because up to 74.3% of the responders relapsed 40 days-44 months after
the end of the treatment period.^184,186-190

            The
data on the evolution of clinically important laboratory parameters, during and
after meglumine antimoniate administration, that can be extracted from these
RCTs are limited: hematocrit increased significantly at the end or 1 month
after the end of treatment,^184,189
albumin concentration increased significantly at the end of treatment,¹⁸⁹ gamma-globulin concentration was within
reference range 5 months after the end of treatment,¹⁸⁹ serum creatinine concentration did not
increase¹⁸⁴ or was higher at the end of 2 weeks after the
end of treatment compared to time 0 in 10-10.8% of the dogs^181,186 or
increased significantly,¹⁸⁸ and the prevalence of proteinuria was
significantly lower at the end of treatment.¹⁸⁴

            A
reduction of parasitic load, based on bone marrow^181,186,190
and/or lymph node microscopy¹⁸⁶ at the end of treatment¹⁸⁶ and after 14,¹⁸¹ 40,¹⁸⁶ 50,¹⁹⁰ 100,¹⁸⁶ or 140 days¹⁹⁰ was a uniform finding in the 3 RCTs where this
parameter was evaluated. Of the dogs with positive microscopy and/or culture
(bone marrow, lymph node, skin) before treatment, 80-100% and 37.5-75% were
negative for the same examinations at the end^184,186 or 1-5 months after the
end of treatment,^186,189,190
respectively. However, in parallel with the clinical relapses after treatment
discontinuation, bone marrow and lymph node qPCR changed from negative at 1
month to positive at 4 months after the end of treatment in 33.3% of the dogs,
at the same time point it was negative in only 16.7% of them,^187,188 and
bone marrow and lymph node parasitic load 5 months after the end of treatment
were not lower than on day 0 anymore.¹⁸⁶ Also, 100% (4/4) of the dogs that survived 5
months after the end of treatment were infectious to sand flies.¹⁸⁹

            Meglumine
antimoniate administration enhanced the parasiticidal activity of macrophages,
and this effect was more pronounced in dogs with lower parasitic burden after
treatment,¹⁹⁰ whereas Leishmania-specific antibody
concentrations remained unchanged,¹⁸⁶ decreased,^181,189 or
initially decreased and then increased,^187,188
depending on the duration of the RCT and the timepoint(s) of evaluation.

            The
prevalence of adverse drug reactions was 9.5-66.7%,^181,184,187 but
none of them was severe.¹⁸⁶ Reported adverse effects include injection
site reactions, sometimes associated with local edema and lameness,^181,184
depression,¹⁸¹ lethargy,¹⁸¹ anorexia,¹⁸¹ weight loss,^181,187
vomiting,¹⁸¹ and diarrhea.¹⁸¹ Additional, not common, adverse effects
reported in non-RCTs include hyperthermia, acute pancreatitis, deterioration of
kidney function, uveitis, arthritis, and leukopenia.^192-196 However, at least for some of them, it is not
clear if they are real adverse drug reactions, or if they appear due to CanL or
due to the massive death of parasites with subsequent release of their antigens
that triggers or exacerbates immune-complex mediated pathologies, such as uveitis
and polyarthritis.

            Finally,
although not examined in these RCTs, it is well established that the widespread
use¹⁹⁷ and the repeated administration of meglumine
antimoniate to the same dog^198,199
promote drug resistance in L. infantum.

            Despite these drawbacks, meglumine
antimoniate monotherapy has been proposed for dogs at LeishVet stage I of CanL.^88,99 This probably stems from dogs with papular
dermatitis, no additional clinical or laboratory abnormalities, and strong Leishmania-specific
cell-mediated immunity, that may respond to this treatment without relapsing or
progressing to a more advanced stage of the disease. However, these dogs may
also self-cure or may respond to topical treatment (see later),²⁰⁰ and, in the absence of RCTs showing an
advantage of parenteral meglumine antimoniate administration, this suggestion
cannot be supported.

Conclusion: Meglumine antimoniate
monotherapy is not indicated for the treatment of CanL because of the frequent
relapses, which may lead to the death of the dog or necessitate repeated
administration that promotes drug resistance (SORT: strong).

            Compared to the free drug, liposomal
formulations of meglumine antimoniate offer the theoretical
advantage of longer half-life and increased antimony concentrations in target
organs, like liver and spleen.^201-206 They have been evaluated in three RCTs (Table
S2).^202,207,208 In
all of them, the drug was administered at the dose of 23 mg/kg (corresponding
to 6.5 mg antimony/kg) IV every 4 days, for a total of four²⁰² or six^207,208
injections. In these studies, liposomal meglumine antimoniate was compared to
allopurinol,²⁰⁸ anti-canine interleukin-10 receptor (IL-10R)
monoclonal antibody,²⁰⁷ placebo,^202,208 and
to liposomal meglumine antimoniate-allopurinol combination.²⁰⁸ The number of dogs treated with liposomal
meglumine antimoniate varied from eight²⁰⁸ to 12,²⁰² the diagnosis of CanL was confirmed by
serology (3/3),^202,207,208 bone
marrow culture (1/3),²⁰⁷ and bone marrow PCR (1/3),²⁰⁸ and the severity of CanL was not reported in
one RCT,²⁰⁷ another study included 1/8 stage I dogs, 2/8
stage II dogs, 3/8 stage III dogs, and 2/8 dogs of unknown stage classified
with a modified LeishVet staging algorithm,²⁰⁸ and the third RCT enrolled four
“asymptomatic”, four “oligosymptomatic” and four “symptomatic” dogs.²⁰² The quality of these studies is intermediate²⁰⁷ or low.^202,208

            The
results of these RCTs on the efficacy of liposomal meglumine antimoniate are
inhomogeneous: in one study, none of the dogs responded, 33.3% were euthanized
due to CanL, and 4.5 months after the end of treatment the clinical score was
higher compared to day 0;²⁰²  in
another study, there was a non-significant decrease of clinical score at 10 and
at 70 days after the end of treatment that was followed by deterioration and, 3
months later, the clinical score was only 2.5% lower than before treatment;²⁰⁷ and in the third study, 62.5% of the dogs
showed clinical improvement at 6 months, whereas 25% had died of unrelated
reasons.²⁰⁸ The evolution of laboratory parameters is
presented in only one RCT,²⁰⁷ where 10 days after the end of treatment PCV
was significantly higher compared to time 0, and there were no significant
changes in platelet count, as well as in total protein, globulin, urea nitrogen
and creatinine concentrations.

            Comparisons
of qPCR results on parasitic load before and after treatment are somehow
inconsistent: at 4 months it was decreased in the bone marrow and spleen,²⁰⁸ at 5 months it was not decreased in the bone
marrow,²⁰⁷ and at 6 months it was decreased in the spleen
and skin, but not in the bone marrow.²⁰⁸ Also, all surviving dogs had positive bone
marrow culture 4.5 months after the end of treatment.²⁰² A reduction of infectivity to L.
longipalpis was recorded in one study: although the decrease of the
prevalence of dogs positive in xenodiagnosis was not significant (from 50%
before treatment to 16.7% at 4 months and to 33.3% at 6 months after the end of
treatment), the percentage of infected sand flies was significantly lower at
both post-treatment time points compared to baseline;²⁰⁸ this finding was further strengthened by the
results of another RCT where the percentage of infected sand flies was
significantly lower among those fed on treated dogs 4.5 months after the end of
treatment, compared to those fed on placebo-treated dogs.²⁰²

            Seventy
days after the end of treatment, in addition to the increased peripheral blood
lymphocyte subpopulations [CD3+, CD4+, interferon-γ (INF-γ)-positive CD4+,
CD8+, and CD21+ cells), Leishmania antigen-induced proliferation of CD4+
cells was significantly higher compared to baseline.²⁰⁷ However, at the same time point, the numbers
of interleukin-4 (IL-4)-positive CD4+ cells were also higher compared to time
0, and 3 months later, IL-10 production by PBMCs after stimulation with Leishmania
antigen increased despite progressive clinical deterioration.²⁰⁷ Also, the levels of Leishmania-specific
IgG 6 months after the end of treatment did not differ from baseline.²⁰⁸

            Although
liposomal meglumine antimoniate is considered safer than the free drug due to
the lower total dose of antimony,²⁰² transient adverse effects occurred in up to
90% of the dogs during and soon after the IV administration, and they included
tachycardia, tachypnea, salivation, vomiting, defecation, urination,
prostration, mydriasis followed by miosis, ataxia, and tremors.^202,207

Conclusion: Liposomal formulations of
meglumine antimoniate monotherapy are not indicated for the treatment of CanL
because of the frequent relapses, which may lead to the death of the dog (SORT:
moderate).

            A RCT that was not included in our
systematic review because it was published after 2022, examined the efficacy
and safety of a topical formulation of meglumine antimoniate (30% in pluronic
F-124), sprayed twice daily for one month over the skin lesions of dogs with
the papular form of CanL, that (with very few exceptions) was not accompanied
by other clinical signs or laboratory abnormalities. The results were promising
because there were no adverse effects and a complete response to treatment was
recorded in 70% of the dogs.²⁰⁰

8.3.2. Miltefosine

            Miltefosine is a repurposed drug
that was initially developed as antineoplastic agent²⁰⁹ and later approved for the treatment of CanL,
and, in some countries, for the treatment of human VL and CL. Miltefosine accumulates
inside macrophages and causes apoptosis-like death of amastigotes through
interference with multiple metabolic pathways, including those responsible for
lipid and ATP synthesis and calcium homeostasis.²¹⁰ Also, it has indirect anti-Leishmania
effects by activating macrophages, T lymphocytes, and Th1-like immune
responses.²¹¹ After oral administration (in the food) at the
registered dose of 2 mg/kg once daily, it is well absorbed and accumulates very
slowly in the body due to the long half-life. The duration of treatment is 4
weeks.

            The
efficacy and safety of miltefosine in dogs with CanL have been evaluated in one
RCT (Table S3) of 6-week duration (drug administration during the first 4 weeks
and 2-week follow-up).¹⁸¹ In this study, miltefosine was compared to
meglumine antimoniate; 46 miltefosine-treated dogs are eligible for the
evaluation of efficacy and 55 for the evaluation of safety; the confirmation of
CanL diagnosis was based on serology and/or bone marrow microscopy and/or bone
marrow PCR, and a serum creatinine concentration within reference range was an
inclusion criterion. The quality of the study is intermediate. At 6 weeks, total
clinical score was significantly decreased (by 51.1%) and in 52.2% of the dogs
it was ≥ 60% lower than baseline; however, 23.3% of the initially enrolled dogs
had been lost to follow-up and ITT analysis was not performed. Considering
laboratory parameters, none of the dogs had higher creatinine concentration at
study conclusion compared to time 0. The parasitic load was probably reduced,
because only 10% of the 30 dogs with positive bone marrow microscopy before
treatment were still positive at the end of the trial. The effect of treatment
on parasite transmission to sand flies or on Leishmania-specific cell
mediated immunity was not evaluated, and a ≥ 2-fold decrease of IFA titer was
recorded in 9.1% of the dogs. The prevalence of adverse drug reactions was
30.9%: they included depression or lethargy (3.6%), anorexia, vomiting and/or
diarrhea (30.9%), and polyuria-polydipsia (1.8%).¹⁸¹

            In
other studies, including one RCT published after 2022²¹² and five open trials,^213-217 the clinical efficacy was variable: 1 month^213,217 and 2 months²¹² after end of treatment, 20% of dogs did not
present clinical signs, and at 23 months, 50% of dogs were considered
clinically cured;²¹⁴ compared to time 0, the clinical score was²¹⁷ or was not²¹² significantly lower at the end of treatment
period, it was significantly lower after 2²¹⁶ and 4 weeks,²¹⁷ and it was²¹⁶ or was not²¹² significantly lower after 2 months; by the end
of treatment period, 90% of dogs showed clinical improvement,²¹⁵ and after 2 weeks 50% of dogs had >75%
reduction of clinical score compared to baseline;^213,217 however, 2 months and 2
years after end of treatment, 20%²¹² and 14.3%,²¹⁴ of dogs, respectively, had died or were euthanized
due to CanL. Of the clinically important laboratory parameters, 1 month after
end of treatment there was no change in hematocrit, platelet count, total protein,
beta-1 globulin, beta-2 globulin, and gamma-globulin concentration,²¹⁷ but albumin concentration and albumin/globulin
ratio were significantly higher compared to time 0.²¹⁷ Short-term data on parasitic load are
conflicting, with some studies showing a reduction,^214-216 but the RCT showing lack of change at the end
of treatment period and 2 months later;²¹² nevertheless, long-term monitoring showed that
parasitic load started to increase.²¹⁵ The percentage of treated dogs that were
infectious to sand flies was decreased 2 months after end of treatment (from
51.4% to 25.7%),²¹⁶ whereas, at the same time point, IFA titer did
not differ significantly from baseline.²¹² Later-on, antibody levels decreased only to
increase again 10 months after end of treatment.²¹⁴

            Induction
of miltefosine-resistant strains of L. infantum is to be expected due to
the long half-life that exposes surviving parasites to subtherapeutic levels of
drug after treatment discontinuation, and it has been confirmed in a dog
treated with the miltefosine-allopurinol combination.²¹⁸

            Like
for meglumine antimoniate, miltefosine monotherapy for dogs at LeishVet stage I
of CanL⁹⁹ cannot be adopted without further studies.

Conclusion: Miltefosine
monotherapy is not indicated for the treatment of CanL because of the limited
data on long-term efficacy along with the conflicting results of non-controlled
trials (SORT: moderate).

8.3.3. Allopurinol

            Allopurinol is parasitostatic for L.
infantum promastigotes and intracellular amastigotes, an effect mediated by
interruption of parasite purine salvage pathway and protein synthesis.^219,220 The recommended dose is 10 mg/kg, orally,
twice daily for at least 6-12 months.²²¹

            The efficacy and safety of
allopurinol have been evaluated in five RCTs (Table S4).^208,221-224 In these studies, allopurinol was compared to
liposomal meglumine antimoniate,²⁰⁸ placebo,^208,221 or no treatment,^222,224 and allopurinol monotherapy was compared to
its’ combination with meglumine antimoniate,²²³ liposomal meglumine antimoniate,^208,224 or a defined subunit vaccine (Leish-F2)
formulated with second generation lipid adjuvant in
stable emulsion (SLA-SE).²²² The daily dose of the drug varied from 20
mg/kg to 60 mg/kg, that were either administered once daily^208,222 or were split and given twice daily^221,223,224 for 60 to 140 days. The number of
allopurinol-treated dogs varied from six^222,223 to 37,²²¹ and the confirmation of CanL diagnosis was
based on serology (4/5)^{208,221,223,224} and/or the demonstration of parasite by
microscopy (1/5)²²³ and/or the demonstration of parasitic DNA in
bone marrow with molecular methods (3/5).^{208,221,222,224} The severity of CanL is reported in two
studies: in one of them CanL were classified as stage I (6.3%), stage II
(56.3%) or stage III (37.5%) using a modification of LeishVet classification
system,²⁰⁸ whereas, in the second study, CanL was
classified as stage I (12.5%), stage II (75%) or stage III (12.5%) using a
clinicopathological scoring system from 0
to IV;²²⁴ in two additional studies, dogs with CanL and
CKD^221,223 or liver insufficiency²²³ were excluded. The quality of these five
studies is intermediate^221-223 or low.^208,224

            Complete clinical cure was rare
(2.7% of dogs after 4-month treatment,²²¹ and 6.3% of dogs 2 months after treatment
discontinuation),²⁰⁸ but clinical improvement was seen after 20
days²²³ and in at least 25% of dogs between the end of
treatment and the following 2 months.²⁰⁸ Also, at the end of a 4-month treatment period
severity of 10 clinical signs of CanL was significantly lower compared to time
0,²²¹ whereas 2 months or 9 months after the end of
a 140-day or a 3-month treatment period, respectively, clinical score was
significantly lower than in the placebo groups.^208,222 However, clinicopathological score on
treatment day 130 did not differ between allopurinol-treated and untreated
dogs,²²⁴ 42.9% of dogs relapsed within a 4-month period
after treatment discontinuation,²²⁴ and 8.1-12.5% of the dogs died due to CanL.^221,224

            Prevalence of some clinicopathologic
abnormalities, such as anemia (after 1 month²²³ and 4 months²²¹ of treatment), hyperproteinemia,
hyperglobulinemia, decreased albumin/globulin ratio, and increased inorganic
phosphorous (after 4 months of treatment),²²¹ was significantly lower than on time 0; on the
contrary, there was no significant change in the prevalence of hypoalbuminemia,
increased BUN, increased creatinine, and proteinuria.²²¹ Allopurinol administration for 2 months
resulted in a significant decrease of total protein, CRP and ceruloplasmin
concentration, and in non-significant changes in albumin, alpha-2 and gamma-2
globulins.²²³ Also, after 130 days of treatment, BUN and
creatinine concentration did not differ from those on day 0.²²⁴

            Data on the efficacy of allopurinol
in terms of parasitic load reduction are conflicting. Semiquantitative microscopic
examination of lymph node and bone marrow smears showed a significantly lower
number of amastigotes after 4 months of treatment but all dogs were bone marrow
PCR-positive.²²¹ The latter was also found after drug
administration for 130 days, based on bone marrow, liver and spleen qPCR and on
skin IHC, but parasitic density was not lower than on day 0.²²⁴ When allopurinol was administered for 140 days
and the results of qPCR were compared between baseline and 2 months after
treatment discontinuation, skin but not bone marrow parasitic density had
decreased significantly.²⁰⁸ Finally, when it was administered for 3
months, and bone marrow qPCR was performed during (day 63) as well as 3 and 9
months after treatment discontinuation, parasitic density was higher at the
latter two time points, and all dogs were qPCR-positive at 9 months.²²² Allopurinol treatment reduces infectivity of
dogs to sand flies: 56.3% (9/16) dogs with CanL were infectious to L.
longipalpis before treatment vs 0% (0/16) at the end of a 140-day
treatment period and 6.3% (1/16) 2 months after treatment discontinuation; the
percentage of infected sand flies was significantly lower at the latter time
point compared to baseline.²⁰⁸

            There
are no data on the effect of allopurinol on Leishmania-specific cell-mediated
immunity and the available information on humoral immunity is inconsistent. In
one study, administration for 4 months resulted in significant reduction of IFA
titers and ELISA ODs, and 5.9% of dogs became negative in both tests.²²¹ On the contrary, in two other studies, reduction of
IFA titers or ELISA ODs was not significant at either the end of allopurinol
administration for 130-140 days or 2-4 months after drug withdrawal.^208,224

            In two
RCT no adverse effects were noticed,^221,222 but in another study, allopurinol administration at much higher than the
recommended dose (30 mg/kg, twice daily) for 130 days resulted in kidney
xanthine depositions in half of the dogs.²²⁴ Xanthinuria, that can also lead to lithiasis in renal
pelvis and/or urinary bladder, is a well-known adverse effect of allopurinol
and may be prevented by feeding a low-protein, low-purine diet for the whole
period of drug administration.²²⁵

            Long-term
allopurinol administration probably promotes the development of resistant
strains of L. infantum due to their positive selection under drug
pressure, and this has been linked to relapses of CanL.¹⁶⁹

            The
recommendation of using allopurinol monotherapy in dogs at LeishVet stage I of
CanL (mainly dogs with papular dermatitis)⁹⁹ cannot be adopted for the same reasons given for
meglumine antimoniate and miltefosine. Due to lack of relevant studies, it is
much more difficult to give evidence-based recommendations in favor⁹⁹ or against allopurinol single agent therapy for dogs
at stage IV of CanL (CKD stage III-IV or extreme proteinuria with or without
thromboembolism). If treatment is attempted despite poor prognosis, the initial
aim should be to halt the deterioration of and to improve kidney function;¹⁷² subsequent anti-Leishmania treatment should be
effective enough to reduce parasitic burden and the deposition of
immune-complexes in the glomeruli, and allopurinol does not seem to fulfil this
criterion.

Conclusion: Allopurinol
monotherapy is not indicated for the treatment of CanL because of the limited
efficacy in terms of clinical improvement and amelioration of clinicopathologic
abnormalities (SORT: strong) and the inconsistent efficacy for the reduction of
parasitic load (SORT: moderate).

8.3.4. Aminosidine (paromomycin)

            Aminosidine
is an aminoglycoside antibiotic with broad antiprotozoal activity that is not
shared with the other drugs of the same class. After binding to 30S ribosomal
subunit of the parasite, it inhibits protein synthesis and subsequently blocks
energy production and alters membrane permeability, leading to death.^226,227 Currently recommended dose is 15 mg/kg, SC, once daily, for at least 3
weeks.^228,229

            The
efficacy and safety of aminosidine have been evaluated in a single RCT (Table
S5),¹⁸⁶ where it was compared to its combination with
meglumine antimoniate and to meglumine antimoniate monotherapy. Eleven dogs
with CanL of unknown severity, confirmed by serology and microscopy or culture,
were treated with aminosidine for 21 days; the quality of the study is
intermediate. Unfortunately, the daily dose of the drug (7 mg/kg, split into
two daily SC injections) was approximately half the recommended one, thus
limiting the relevance of the results.

            Ten of
the 11 dogs (90.9%) responded to the treatment, and their response was
considered complete (1/10), good (5/10), or moderate (4/10). However, 8/10
(80%) relapsed within approximately 40 (4/8) or 160 (4/8) days after treatment
discontinuation. Bone marrow microscopy showed a significant reduction of
parasitic load at the end of treatment, and after 40 and 100 days compared to
day 0, but the significance was lost at 160 days, and there was no similar
change in lymph nodes. In total, both bone marrow and lymph node microscopy
were negative for Leishmania amastigotes in 54.5% (6/11) of the dogs at
the end of treatment and after 40 days, in 45.5% (5/11) after 100 days, and in
only 18.2% (2/11) after 160 days, and there were no significant changes in IFA
titer at any of the above time points. There were no serious adverse effects
and only 1/11 (9.1%) dogs had a transient increase in BUN and creatinine
concentrations,¹⁸⁶ which is similar to the results of an open study using
the recommended dose (15 mg/kg, SC, once daily) of the drug.²²⁹

            No
information on the evolution of important clinicopathologic abnormalities, the
infectivity to sand flies or the possible changes in cell-mediated immune
response against the parasite, during and after treatment, is provided by the
RCT. However, an open trial using the recommended dose of aminosidine showed a
significant increase of hematocrit and Hb 3 months after the end of treatment,²²⁹ and another study using a lower dose (5 mg/kg, SC,
twice daily, for 28 days) showed a tendency for increased albumin/globulin ratio
and decreased proteinuria.²³⁰

            There
are no studies on the induction of resistant strains of the parasite during
aminosidine treatment, but there are some data supporting that
antimony-resistant L. infantum can show cross-resistance to aminosidine.¹⁹⁹ Finally, being an aminoglycoside, aminosidine may
promote bacterial resistance.

Conclusion: Aminosidine
(paromomycin) is not indicated for the treatment of CanL because of the
relapses after treatment discontinuation (SORT: moderate).

8.3.5. Marbofloxacin

            Marbofloxacin
is a 3^rd generation fluoroquinolone that inhibits Leishmania
topoisomerases and subsequently interferes with the replication of parasite
DNA. Moreover, it increases tumor necrosis factor-a (TNF-a) and nitrogen dioxide
production by infected macrophages.²³¹ The recommended dosage regimen for the treatment of CanL
is 2 mg/kg orally once daily for 10-40 (usually 20-28) days.²³²

            The
efficacy and safety of marbofloxacin have been evaluated in one RCT (Table S6),²³² where a comparison was made among different treatment
durations (10, 20, 28, and 40 days), at the recommended daily dose of 2 mg/kg.
Twenty-four dogs with CanL of unknown severity, confirmed by lymph node
microscopy and culture, were included, and the quality of the study is
intermediate.

            Twelve
weeks after treatment initiation 66.7% (16/24) of the dogs were considered
clinically cured, and 8.3% (2/24) improved. However, five dogs relapsed within
9 months from the beginning of treatment. Lymph node microscopy showed a
significantly lower parasitic density at 12 weeks compared to time 0, and none
of the dogs presented adverse effects.²³²

            No
information on the evolution of important clinicopathologic abnormalities, the
infectivity to sand flies, or the possible changes in cell-mediated and humoral
immune responses against the parasite is provided by the RCT. However, in an
open trial of dogs with CanL and CKD [International Renal Interest Society
(IRIS) stage I (39.3%), II (21.4%), III (28.6%) or IV (10.7%)] there were no
significant changes in hematocrit, BUN, creatinine, inorganic phosphorus and UPC,
whereas albumins increased and globulins decreased at the end of the 4-week
treatment period.²³³ In another open trial where 61 dogs with CanL received
marbofloxacin for 4 weeks, ELISA ODs did not differ between time 0 and 3 months
later.²¹³ Finally, being a fluoroquinolone, marbofloxacin can
promote bacterial resistance and for this reason it is considered a 2^nd
tier antimicrobial.²³⁴ 

Conclusion: Marbofloxacin
is not indicated for the treatment of CanL because of the relapses after
treatment discontinuation (SORT: moderate), the paucity of information on
critical features of this treatment, such as the effect on infectivity to sand
flies and on parasite-specific cell-mediated immunity, and the risk to induce
bacterial resistance (SORT: weak). Administration of marbofloxacin should be
considered in dogs with CanL and concurrent bacterial infections if the
responsible organisms are resistant to 1^st tier antibacterials and
susceptible to marbofloxacin, and perhaps in dogs with CanL and CKD IRIS stage
III or IV (SORT: weak).

8.3.6. Metronidazole

            After
enzymatic activation, metronidazole produces toxic metabolites causing damage
to Leishmania DNA. The recommended dose is 25 mg/kg orally once daily
for 90 days, and the veterinary product that has been administered to dogs with
CanL contains also spiramycin (150,000 UI/kg orally once daily).²³⁵

One RCT (Table S7)²³⁵ compared the efficacy and safety of metronidazole
(with spiramycin) in 13 dogs with CanL of unknown severity, with that of
meglumine antimoniate-allopurinol combination. The diagnosis of CanL was
confirmed by serology, microscopy and/or PCR, and the quality of the study is
intermediate.

By the end of the 3-month treatment period,
the clinicopathological score was significantly lower compared to day 0;
specifically, it was decreased in 83.3% (10/12) of the dogs, increased in 16.7%
(2/12), and one dog had been removed from the study because it developed
pemphigus foliaceus. Clinical improvement occurred after 15-45 (median 15)
days, but in 33.3% (3/9) of dogs it was followed by an increase of
clinicopathological score, beginning at 30-60 days after treatment
discontinuation. The only available specific information on the evolution of
clinicopathologic abnormalities is that BUN increased in 7.7% (1/13) of the
dogs. Four months after treatment discontinuation, bone marrow, lymph node
and/or blood PCR was positive in all dogs and ELISA ODs were like those before
treatment. Apart from the dog with pemphigus foliaceus, no other adverse
effects were recorded. No data are available on the infectivity of treated dogs
to sand flies or possible changes of their Leishmania-specific
cell-mediated immunity.²³⁵ 

Conclusion: Metronidazole
is not indicated for the treatment of CanL because of the frequent relapses
after treatment discontinuation (SORT: moderate) and the limited information on
some critical features of this treatment, such as the evolution of
clinicopathologic abnormalities, infectivity to sand flies and Leishmania-specific
cell-mediated immunity; moreover, concerns arise from the long-term
administration of an antibacterial agent (SORT: weak), as for marbofloxacin.

8.3.7. O-alkyl-hydroxamate
(MTC-305)

            It is a vorinostat derivative that inhibits histone
deacetylases. It has been administered at 3.75 mg/kg SC once daily in two
cycles, each with a 4-week duration, that were separated by 1 month without
treatment, and has been evaluated in a single RCT (Table S8).¹⁸⁷ In that study, it was given to six dogs with CanL
(LeishVet stage I-III), the diagnosis of which was confirmed by serology and
qPCR, and was compared to meglumine antimoniate monotherapy and to its
combination with meglumine antimoniate. The quality of the study is intermediate.

            At 7 months (4 months after the end
of the 3-month treatment period) none of the dogs had a clinical score of zero.
However, clinical improvement occurred between 2 and 4 months, and by 7 months
the clinical score decreased by 64.7% and was lower than baseline in 83.3%
(5/6) dogs. On the other hand, half of the dogs had higher clinical scores at 7
months compared to 3 months, indicative of possible relapse after treatment
discontinuation. A similar trend was observed for the parasitic density that
was measured in bone marrow, lymph nodes and blood by qPCR: at 7 months it was
significantly lower compared to day 0 (bone marrow, lymph nodes), and qPCR was
negative in 1/6 (bone marrow) or 2/6 (blood) dogs. Between 3- and 7- months it
increased in 2/6 (bone marrow, blood) or 5/6 (lymph nodes) dogs. Total Leishmania-specific
IgG IFA titers remained stable, and the only adverse effect was a decreased
neutrophil count at 3 months. Possible changes in cell-mediated immune
responses were evaluated with a non-validated approach (measurement of
parasite-specific IgG subclasses and of INF-γ and IL-4 mRNA in blood), and
there are no data on the evolution of clinicopathologic abnormalities and the
infectivity of treated dogs to sand flies.

Conclusion: O-alkyl-hydroxamate
(MTC-305) is not indicated for the treatment of CanL because of the incomplete
clinical response, the tendency for relapse after treatment discontinuation
(SORT: moderate) and the limited information on some critical features of this
treatment, such as the evolution of clinicopathologic abnormalities,
infectivity to sand flies and Leishmania-specific cell-mediated immunity.

8.3.8. (-)-α-bisabolol

            This
molecule is a sesquiterpene causing mitochondrial damage and perhaps apoptosis
to Leishmania.  It has been
administered at 30 mg/kg orally once daily in two cycles, each with a 4-week
duration, that were separated by 1 month without treatment, and has been
evaluated in a single RCT (Table S9).¹⁸⁸ In that study, it was given to six dogs with CanL of
clinical severity that is hard to determine, the diagnosis of which was
confirmed by serology and blood qPCR, and was compared to meglumine
antimoniate. The quality of the study is intermediate.

            Only
half of the dogs (3/6) completed the trial, and ITT statistical analysis was
not performed, making interpretation of the results difficult. At 7 months (4
months after the end of the 3-month treatment period) none of the dogs was
clinically cured. However, clinical improvement occurred between 2 and 4 months,
and by 7 months the clinical score had decreased by 33.3% and was lower than
baseline in all three dogs. On the other hand, 1/3 dogs had higher clinical
score at 7 compared to 4 months, indicative of possible relapse. By 7 months,
hematocrit was increased by 7.1% but platelet count was decreased by 50.8%,
total proteins and globulins were increased by 13% and 24.3%, respectively,
albumins and albumin/globulin ratio were decreased by 3% and 30%, respectively,
and BUN and creatinine were increased by 36.9% and 70.5%, respectively.
Parasitic density was measured in bone marrow, lymph nodes and blood by qPCR
but their changes are not reported; nevertheless, at 7 months each one of the
three tissue samples was qPCR negative in 1/3 dogs, but bone marrow (1/3 dogs)
and blood (2/3 dogs) parasitic density was higher at 7 compared to 4 months.
Parasite-specific IgG IFA titers remained stable, and no adverse effect was
recorded. Possible changes in cell-mediated immune responses were evaluated
with a non-validated approach (measurement of INF-γ and IL-4 mRNA in blood) and
there are no data on the infectivity of treated dogs to sand flies.

Conclusion:
(-)-α-bisabolol is not indicated for the treatment of CanL because of the
incomplete clinical response, the deterioration of important clinicopathologic
abnormalities (SORT: moderate) and the lack of information on some critical
features of this treatment such as the evolution of infectivity to sand flies
and Leishmania-specific cell-mediated immunity

8.3.9. Artesunate

            This
molecule, which causes apoptosis of Leishmania, has been isolated from
extracts of the Artemisia annua plant. In a single RCT (Table
S10),²³⁶ it was administered at 25 mg/kg orally once daily for
6 days to 16 dogs with CanL of unknown (most likely mild) severity, that had
been confirmed by serology or blood qPCR. In that study, which is of low
quality, the efficacy and safety of artesunate were compared to those of
meglumine antimoniate-allopurinol combination. After 6 months, 13.3% of the
dogs had died of CanL, 46.7% were clinically improved, and 26.7% were
clinically cured; death occurred between the 2^nd and 3^rd
month, clinical improvement was first noticed at 1 month and clinical cure at 3
months. No data on the evolution of clinicopathologic abnormalities or the
infectivity to sand flies are provided, but at the end of the study 80% of dogs
with positive blood qPCR at time 0 became negative, whereas the reverse was not
seen. Leishmania-specific cell-mediated immunity was not studied but the
humoral immune response declined, with a significant reduction of IFA titer
starting at 1 month and resulting in 58.3% of the dogs being seronegative at 6
months. No adverse effects were recorded.

Conclusion: Artesunate
is not indicated for the treatment of CanL because of the moderate rate of
clinical response, the risk of death from CanL (SORT: moderate) and the lack of
information on some critical features of this treatment such as the evolution
of clinicopathologic abnormalities, infectivity to sand flies and Leishmania-specific
cell-mediated immunity.

8.3.10. Combinations of drugs with direct
anti-Leishmania activity

            Since
none of the above drugs is effective for both short-term and long-term
treatment of CanL, their combinations have been explored. At least in theory,
combining drugs with different mechanism of anti-Leishmania activity can
increase efficacy and prevent relapses of the disease.

8.3.10.1. Meglumine antimoniate-allopurinol combination

            The
efficacy and safety of meglumine antimoniate-allopurinol combination have been
evaluated in 11 RCTs (Table S11).^{182,223,235-243} In these studies, this combination was compared to allopurinol,²²³ metronidazole (plus spiramycin),²³⁵ artesunate,²³⁶ and to a nutritional supplement with antioxidant
properties (DiLsh^TM; Dynamopet, Italy)²³⁷ monotherapies. Also it was compared to meglumine
antimoniate combinations with allopurinol plus domperidone²⁴³ or plus deslorelin,²⁴² with metronidazole (plus spiramycin),²³⁸ and with a dietary supplement containing nucleotides
and an AHCC (Impromune^®; Bioiberica S.A.U., Spain),²⁴¹ and to the miltefosine-allopurinol,²⁴⁰ and allopurinol-aminosidine^182,239 combinations. In eight studies the daily dose of meglumine antimoniate was
100 mg/kg and the duration of treatment varied from 20 to 28 days,^{182,223,236,239-243} whereas in two studies it was administered at a higher daily dose (110-200
mg/kg) and for a longer period (30-90 days),^235,238 and in one study it was under-dosed (40 mg/kg/day) for 1 month.²³⁷ The daily dose of allopurinol varied from 20 to 40
mg/kg, starting simultaneously with meglumine antimoniate and continuing for a
total treatment period of 3 weeks to 7 months.^{182,223,235-243} The number of dogs varied from six²²³ to 38,²⁴¹ the confirmation of CanL diagnosis was based on
serology (11/11),^{182,223,235-243} and on demonstration of parasite or parasitic DNA by microscopy (8/11)^{182,223,235,238,239,241-243} and/or molecular methods (8/11).^{235-238,240-243} The severity of CanL is reported only in two studies that included dogs at
LeishVet stages II (6/12) and III (6/12)²⁴² or at Canine Leishmaniosis Working Group (CLWG) classification
system stage C,²⁴³ whereas in another study serum creatinine
concentration <1.2 mg/dl and absence of “liver disease” was an inclusion
criterion,²²³ and in three studies dogs with CKD IRIS stage III-IV
were excluded.^182,238,239 The quality of these studies is high,¹⁸² intermediate,^{223,235,237,239-243} or low.^236,238

            According to one of these studies
where a very stringent clinical scoring system was used, 52.6% (10/19) dogs had
absolutely no clinical signs at the end of the trial (day 180),¹⁸² whereas in another RCT clinical cure, first
recorded at 1 month, was present in only 18.2% (4/22) dogs at 6 months;²³⁶ this difference is easily explained because in
the latter study allopurinol was administered only for 1 month, whereas in the
former it was given for the entire 6-month period. Clinical improvement,
starting between 14 and 30 days,^{223,235,236,238,243} was seen in 80% (8/10) of the dogs at 3
months,²³⁵ and in 54.5% (12/22, in addition to the 4/22
clinically cured) dogs at 6 months despite short-term (1 month) allopurinol
administration.²³⁶ All studies reporting total clinical score
and/or number of clinical signs per dog and/or severity of clinical signs found
them to be significantly lower at the end compared to day 0.^{182,235,237,240-242} Death due to CanL, treatment adverse effects
or irrelevant reasons was uncommon [0% (0/6, 0/10, 0/12, 0/14, 0/15 or 0/18),^{223,235,237,238,242,243} 5% (1/20),^182,239 5.3% (2/38),²⁴¹ or 8.3% (3/36)²⁴⁰] except in the RCT where allopurinol was
administered for only 1 month: 5 months later, 23.1% (6/26) of the dogs had
died (2/6 due to CanL).²³⁶ Relapses after treatment discontinuation were
studied in only one RCT where treatment was administered for 3 months: clinical
score started to increase after 30-60 days and after 4 months 25% (2/8) of the
dogs had a relapse, with their clinical score being higher than on day 0.²³⁵ In open long-term trials relapses are also reported,
despite continued administration of allopurinol for years.²⁴⁴

            Starting from the first 10-30 days
of treatment, improvement or amelioration of clinically important
clinicopathologic abnormalities, including anemia,^182,223 hyperproteinemia,^182,223,241 hypoalbuminemia,^182,237,241 hyperglobulinemia,^182,238 increased gamma-globulin concentration,^237,240,241 decreased albumin/globulin ratio,^182,240 and increased concentration of positive acute
phase proteins like CRP,^182,223,241 ferritin,²⁴¹ and ceruloplasmin²²³ was an almost uniform finding. The only
exceptions were lack of change in protein electrophoresis abnormalities and CRP
concentration after 3 weeks of treatment,²⁴³ and lack of change in total protein, albumin,
alpha2-globulin, and gamma2-globulin concentrations recorded in the RCT where
meglumine antimoniate was under-dosed,²³⁷ and in two RCTs where allopurinol was
administered for only 50 days²²³ or 3 months.²³⁸ There was no evidence of deterioration in
kidney function: BUN^{182,237,239,241} and creatinine^237,240,241 concentrations remained stable, inorganic
phosphorus concentration decreased,¹⁸² prevalence of proteinuria did not change²³⁹ or was decreased,²⁴⁰ and UPC values decreased.^239,241 In two RCTs there was increased creatinine
concentration at 2 or 6 months, but it was attributed to increased muscle mass,
because it was not accompanied by parallel changes in BUN or inorganic
phosphorous concentrations, the prevalence of proteinuria or UPC values.^182,239 The lack of nephrotoxicity is further
supported by the results of an open trial that included dogs at LeishVet stage
II or III of CanL with CKD IRIS stage I or II, where, in addition to the
classical markers of kidney function, a stable glomerular filtration rate was
shown during meglumine antimoniate-allopurinol plus symptomatic treatment for
CKD.²⁴⁵

            An early (starting from the first
month) and sustained reduction of parasitic load, based on bone marrow and/or
lymph node microscopy¹⁸² and qPCR^182,240,241 was found. Of the initially positive dogs, at
the end of 6-month treatment period, 36.8% became negative on bone marrow and
lymph node microscopy and bone marrow qPCR.¹⁸² Four months after the end of 90-day treatment
period 25% became bone marrow, lymph node, and blood PCR-negative,²³⁵ and 5 months after the end of 30-day treatment
period 57.1% became blood qPCR-negative.²³⁶ Infectiousness to sand flies was not evaluated
in these 10 RCTs; however, an observational study showed that all eight
initially positive dogs became negative on xenodiagnosis after 6 months of
treatment.¹⁶⁶

            Meglumine antimoniate and
allopurinol combination treatment induced Leishmania-specific
cell-mediated immunity, exemplified by the increased prevalence of positive
leishmanin skin test at the end of 6-month treatment period (73.3%) compared to
day 0 (31.6%),¹⁸² whereas the significant increase of CD4+,
along with the non-significant increase of CD8+ lymphocytes, at 6 months may
denote restoration of the non-specific cell-mediated immune defects of CanL.²⁴¹ With the exception of one short-term (90 day)
RCT,²³⁵ an early (starting at 1-3 months) reduction of
Leishmania-specific antibody concentrations in serum, that was
significant at the end of treatment, was a uniform finding.^{182,236,237,240-242} Also, 57.9% of initially seropositive dogs
became seronegative at 6 months,¹⁸² and 0-29.4% remained seronegative 4-9 months
after treatment discontinuation;^235,236,238 however, in one, short-term, RCT antibody
concentration increased after allopurinol withdrawal in 50% of the dogs.²³⁵

            Adverse effects were seen in 0-60%
of the dogs,^{182,237,238,243} and included injection site reactions^182,235,236
sometimes necessitating meglumine antimoniate discontinuation,²³⁵ “asthenia”,²⁴⁰ vomiting,²⁴⁰ acute pancreatitis causing death (5%),^182,239 possible cutaneous drug eruption,²³⁵ biochemical evidence of hepatotoxicity,²³⁵ and xanthinuria.²⁴¹ It is also logical to anticipate some
additional adverse effects that have already been reported in the RCTs on
meglumine antimoniate (depression, lethargy, anorexia, weight loss, diarrhea)
and on allopurinol (renal mineralization) monotherapies, as well as those
reported in non-RCTs evaluating their combination (xanthine lithiasis).²⁴⁶

            Finally, although not examined in
these RCTs, it is logical to assume that the repeated administration of
meglumine antimoniate and the long-term administration of allopurinol promote
drug resistance in L. infantum, like when these drugs are used as
monotherapies.

Conclusion: Meglumine
antimoniate-allopurinol combination is indicated for the treatment of CanL due
to the consistent, albeit of limited quality, LoE showing that it results in
clinical improvement or cure, in amelioration of clinically important
clinicopathologic abnormalities, in reduction of parasitic load and of
infectivity to sand flies, in upregulation of parasite-specific cell mediated
immunity with downregulation of humoral immunity in most dogs, and that it is
reasonably safe and non-nephrotoxic (SORT: strong). Meglumine antimoniate daily
recommended dose is 100 mg/kg SC, administered either once daily or divided
every 12 hours, for 28-30 days, because lower doses may be less effective and
higher doses or longer treatment periods may increase toxicity without offering
obvious therapeutic benefits (SORT: moderate). Allopurinol dose should be 10
mg/kg orally twice daily for at least 6 months, because higher doses may
increase the frequency of adverse effects (SORT: weak) and shortened treatment
periods are associated with clinical relapses (SORT: strong). Close monitoring
for adverse effects, especially during the first month, is necessary (SORT:
strong). Repeated administration of meglumine antimoniate and unnecessary
extended allopurinol administration periods should be avoided due to the risk
of induction of resistant strains of L. infantum (SORT: moderate).      

            The
use of liposomal formulations of meglumine antimoniate in
combination with allopurinol has been evaluated in two RCTs (Table S12).^208,224 Conventional^208,224 and a combination of conventional and polyethylene glycol (PEG)-containing
(PEGylated)²²⁴ liposomes were used as carrier of meglumine
antimoniate that was administered at the dose of 23 mg/kg (corresponding to 6.5
mg antimony/kg) IV every 4 days, for six times;^208,224 in one study a second treatment “cycle” was given after a 40-day
discontinuation period.²²⁴ In the first RCT allopurinol was administered at a
daily dose of 20 mg/kg for 140 days,²⁰⁸ whereas in the second study a much higher than the
usual daily dose (60 mg/kg) was given for 130 days.²²⁴ In these studies, the two types of liposomes
(conventional or conventional/PEGylated combination) were compared to each
other,²²⁴ and the liposomal meglumine antimoniate-allopurinol
combination was compared to liposomal meglumine antimoniate monotherapy,²⁰⁸ allopurinol monotherapy,^208,224 and to placebo or no treatment.^208,224 The number of treated dogs was eight²⁰⁸ or nine,²²⁴ the diagnosis of CanL was confirmed by serology and
bone marrow PCR, and all dogs had stage II or III CanL based on a modified
LeishVet staging algorithm²⁰⁸ or an unspecified staging system.²²⁴ The quality of both studies is low.

            Of the
eight dogs treated with meglumine antimoniate in conventional liposomes plus
allopurinol at the usually recommended dose, 25% (2/8) died of unrelated causes.
All the remaining dogs responded, their clinical signs were significantly
improved at the end of treatment and 2 months later compared to baseline, and 2
months after allopurinol discontinuation 50% (3/6) were considered clinically
cured. None of the remaining 3 dogs showed evidence of relapse.²⁰⁸ The only useful data on clinical response that can be
extracted from the second RCT are that at the end of allopurinol administration
period (day 130) and 4 months later dogs treated with the combination of
conventional and PEGylated liposome-encapsulated meglumine antimoniate plus
allopurinol had significantly lower clinical score in comparison to the no
treatment group, and that 4 months after allopurinol discontinuation they had a
significantly lower score compared to the group treated with conventional
liposome-encapsulated meglumine antimoniate plus allopurinol.²²⁴ Also, although not clearly stated in the manuscript,
it seems that the percentage of dogs with clinical relapse within 4 months
after treatment discontinuation was 25% and 50% for the conventional plus
PEGylated liposome and the conventional liposome groups, respectively.²²⁴ The evolution of clinicopathologic abnormalities is
not reported, except that there were no changes in BUN or creatinine
concentrations suggesting lack of overt nephrotoxicity.²²⁴

            Bone
marrow, spleen, liver, and/or skin qPCR showed reduced parasitic load at the
end of treatment²²⁴ and 2²⁰⁸ or 4²²⁴ months later compared to time 0. The results regarding
the parasitological-negative dogs after allopurinol discontinuation are
inhomogeneous. In one RCT, 50% (3/6) were negative (bone marrow, spleen, liver,
and skin qPCR, plus bone marrow culture) at 2 months,²⁰⁸ whereas in the other RCT none of the 18 dogs was
negative (bone marrow, spleen and liver qPCR, plus skin IHC) at 4 months.²²⁴ A reduction of infectivity to L. longipalpis
was recorded in one study: 50% (3/6) dogs were positive on xenodiagnosis on day
0 and none of them at the end of the treatment or 2 months later.²⁰⁸

            No
data on the effects of treatment on cell-mediated immunity are available, and
the results about the levels of Leishmania-specific IgG are
controversial. Two months after the end of treatment with conventional
liposome-encapsulated meglumine antimoniate plus allopurinol at the usual dose
there was a significant reduction of IFA titers and 33.3% (2/6) of the dogs
became seronegative. At the end and 4 months after the end of treatment with
conventional liposome-encapsulated meglumine antimoniate plus allopurinol at
the high dose, there were no significant changes in IFA titers or ELISA ODs
compared to time 0.²²⁴ At the end and 4 months after the end of treatment
with conventional/PEGylated liposome-encapsulated meglumine antimoniate plus
allopurinol at the high dose, there were no significant changes in IFA titers
but ELISA ODs were significantly lower than at baseline.²²⁴

            In one RCT, temporary IV
infusion-related adverse effects (salivation, vomiting, defecation) occurred in
all dogs and xanthine nephrolithiasis in half of them, probably due to the high
daily dose of allopurinol.²²⁴

Conclusion: Despite some evidence of
limited quality for efficacy, liposomal meglumine antimoniate-allopurinol
combination cannot be recommended for the treatment of CanL, due to the
inconsistency of the results, the lack of information on some critical features
of this treatment such as the evolution of clinicopathologic abnormalities and Leishmania-specific
cell-mediated immunity (SORT: moderate), and the lack of head-to-head
comparison with conventional meglumine antimoniate plus allopurinol combination
treatment (SORT: weak).

8.3.10.2. Meglumine antimoniate-aminosidine combination

            In
addition to direct anti-Leishmania activity, co-administration of
aminosidine with meglumine antimoniate may modify the pharmacokinetics of the
latter by increasing its persistence in blood.²⁴⁷ The efficacy and safety of meglumine
antimoniate-aminosidine combination were evaluated in a single RCT (Table S13),¹⁸⁶ where it was compared to meglumine antimoniate and to
aminosidine monotherapies. Eleven dogs with CanL of unknown severity, confirmed
by serology and microscopy or culture, were treated with a typical dose of
meglumine antimoniate (106 mg/kg, once daily, SC) and a low dose of aminosidine
(3.5 mg/kg, twice daily, SC) for 21 days; the quality of the study is
intermediate.

            Ten of
the 11 dogs (90.9%) responded to the treatment, and their response was
considered complete (3/10), good (6/10), or moderate (1/10). However, 5/10 (50%)
relapsed within approximately 40 (1/5) or 160 (4/5) days after treatment
discontinuation. Bone marrow and lymph node microscopy showed a significant
reduction of parasitic load at the end of treatment and after 40, 100, and
(lymph node microscopy only) 160 days. In total, both bone marrow and lymph
node microscopy were negative for Leishmania amastigotes in 72.7% (8/11)
of the dogs at the end of treatment and after 40 days, in 63.6% (7/11) after
100 days, and in 45.5% (5/11) after 160 days. IFA titer was significantly lower compared to baseline at the end of
treatment and after 40 and 100 days, but not after 160 days and none of the
dogs became seronegative. There were no serious adverse effects and only 1/11
(9.1%) dogs had a transient increase in BUN and creatinine concentrations.¹⁸⁶

            No
information on the evolution of important clinicopathologic abnormalities, the
infectivity to sand flies, the possible changes in cell-mediated immune
responses, and the induction of resistant strains of the parasite is provided
by this RCT. Theoretically, the combination of these drugs may prevent the
development of parasite resistance.²⁴⁸ However, if resistance will develop against one of the
two drugs, it may also involve the other one due to cross-resistance.¹⁹⁹ Also, being an aminoglycoside, aminosidine may promote
bacterial resistance.

Conclusion: Meglumine
antimoniate-aminosidine combination is not indicated for the treatment of CanL
because of the relapses after treatment discontinuation (SORT: moderate).

8.3.10.3. Meglumine antimoniate-metronidazole
combination

             One RCT (Table S14)²³⁸ compared the efficacy and safety of meglumine
antimoniate-metronidazole (with spiramycin) combination with meglumine
antimoniate-allopurinol combination in 14 dogs with CanL of unknown severity (a
creatinine serum concentration >2 mg/ml was an exclusion criterion). Meglumine
antimoniate was administered at 55-100 mg/kg SC twice daily for 30 or 60 days,
and metronidazole (plus spiramycin) at 25 mg/kg (plus 150,000 IU/kg) orally once
daily for 90 days. The diagnosis of CanL was confirmed by serology, microscopy
and/or PCR, and the quality of the study is low.

There was a significant clinical
improvement, first witnessed at 30 days, and none of the dogs died until the
end of the study, but the rate of clinical cure, clinical improvement without
cure and clinical relapse after treatment discontinuation is not reported. The
only available information on the evolution of clinicopathologic abnormalities
is that albumin and globulin concentrations increased and decreased,
respectively, in dogs treated with meglumine antimoniate for 60 dogs but not in
those treated for 30 days. The evolution of parasitic load and possible changes
in cell-mediated immunity are unknown, whereas 9 months after treatment
discontinuation 50% (7/14) dogs were seronegative and no adverse effects are reported.²³⁸ No data are available on the infectivity of treated
dogs to sand flies.²³⁸ 

Conclusion: Meglumine
antimoniate-metronidazole combination is not indicated for the treatment of
CanL because of the limited information on some critical features of this
treatment such as the evolution of parasitic load, infectivity to sand flies
and Leishmania-specific cell-mediated immunity, the lack of any obvious
benefit compared to the standard meglumine antimoniate-allopurinol combination
(SORT: moderate), and the long-term administration of an antibacterial agent
(SORT: weak).

8.3.10.4. Meglumine antimoniate-O-alkyl-hydroxamate
(MTC-305) combination

            This
combination has been evaluated in a single RCT (Table S15),¹⁸⁷ where meglumine antimoniate and O-alkyl-hydroxamate
were administered at 104 mg/kg and at 1.5 mg/kg, respectively, SC once daily in
two 4-week cycles separated by 1 month without treatment. In that study, the
combination treatment was compared to meglumine antimoniate and to O-alkyl-hydroxamate
monotherapies and administered to six dogs with CanL (LeishVet stages I-III),
the diagnosis of which was confirmed by serology and qPCR. The quality of the
study is intermediate.

            At 7
months (4 months after the end of the 3-month treatment period), 1/6 (16.7%)
dogs had zero clinical score (clinically cured) and then remaining 5/6 (83.3%)
had lower clinical scores compared to day 0. Clinical improvement occurred
between 2 and 4 months, and by 7 months the clinical score had decrease by
62.5%. On the other hand, 2/6 (33.3%) dogs had higher clinical scores at 7
months compared to 3 months, indicative of possible relapse after treatment
discontinuation. A similar trend was observed for the parasitic density that
was measured in bone marrow, lymph nodes and blood by qPCR: at 7 months, blood
qPCR was negative in 2/6 (33.3%) dogs, and between 3- and 7- months it
increased in 3/6 (bone marrow, lymph nodes) or 1/6 (blood) dogs. Total Leishmania-specific
IgG IFA titers were lower at 7 months compared to time 0 in 5/6 (83.3%) dogs,
with 2/6 (33.3%) being seronegative, but 1/6 (16.7%) had higher titer than at 3
months. Weight loss during the first 2 months of treatment was recorded in 2/6
(33.3%) dogs. Possible changes in cell-mediated immune responses were evaluated
with a non-validated approach (measurement of parasite-specific IgG subclasses
and INF-γ and IL-4 mRNA in blood), and there are no data on the evolution of
clinicopathologic abnormalities, or the infectivity of treated dogs to sand
flies.  

Conclusion:
Meglumine antimoniate plus O-alkyl-hydroxamate (MTC-305) combination is
not indicated for the treatment of CanL because of the tendency for relapse
after treatment discontinuation (SORT: moderate) and the limited information on
some critical features of this treatment such as the evolution of
clinicopathologic abnormalities, infectivity to sand flies and Leishmania-specific
cell-mediated immunity.

8.3.10.5. Miltefosine-allopurinol combination

            The
efficacy and safety of this combination in dogs with CanL have been evaluated
in two RCTs (Table S16) of 6²⁴⁹ or 7²⁴⁰ month-duration. In one study, the registered dose of
miltefosine (2 mg/kg orally once daily for 28 days) plus the usual dose of
allopurinol (10 mg/kg orally twice daily for 7 months) was compared to
meglumine antimoniate-allopurinol combination,²⁴⁰ or two dosage regimens of miltefosine (the registered one
for 30 days and a modified one, starting at 1.2 mg/kg once daily for the first
5 days and followed by 2.5 mg/kg once daily for 25 days). Both protocols included
combination with allopurinol (10 mg/kg orally twice daily for 6 months), and were
compared to each other.²⁴⁹ The number of treated dogs varied from 16²⁴⁹ to 37,²⁴⁰ the diagnosis of CanL was confirmed by serology and
either lymph node microscopy²⁴⁹ or bone marrow PCR,²⁴⁰ and the severity of the disease is specified in one
study, where dogs at LeishVet stages II or III were included.²⁴⁹ The quality of both studies is intermediate.

            Neither
study specifies the percentage of clinically cured and/or improved dogs;
however, in both RCTs, starting from 2-3 months, there was a significant
improvement of total clinical score that, at the end of treatment period was
lower than on baseline by 61.7%,²⁴⁹ 71.6%,²⁴⁹ or 89.9%.²⁴⁰ None of the dogs died or was euthanized due to CanL,
but an unspecified number of clinical relapses, despite continued allopurinol
administration, is reported in one RCT in dogs that were treated with the
registered miltefosine dosage regimen;²⁴⁹ the same observation is found in open long-term
trials.²⁴⁴

            Of the
clinically important laboratory parameters, PCV was significantly increased at
1 month,²⁴⁹ γ-globulin concentrations at the end of the study were
within reference interval in 47.8% of the dogs,²⁴⁰ and albumin/globulin ratio started to increase at 3
months and by 7 months had normalized in 25.9% of the dogs.²⁴⁰ There were no changes in creatinine concentrations
throughout the study²⁴⁰ or in UPC at 1 and 2 months,²⁴⁹ and the latter parameter normalized by the end of the
study in 27.8% of the dogs with initially abnormal values.²⁴⁰

            Based
on qPCR, the results on bone marrow parasitic load are discordant: in one RCT
there was no difference between day 0 and day 60,²⁴⁹ whereas in the second study there was a significant
overtime reduction of the amount of Leishmania DNA, starting on day 28.²⁴⁰ In the first study, the prevalence of dogs that
converted from positive (day 0) to negative (day 60) bone marrow qPCR was 14.3%
(2/14) for those treated with the registered miltefosine dosage regimen and 50%
(7/14) for those treated with the modified regimen, whereas the relevant
figures for lymph node microscopy were 94.4% (17/18) and 93.8% (13/16),
respectively.²⁴⁹ In general, the results of non-controlled trials are in
favor of a significant reduction of parasitic load in lymph nodes and/or blood
during long-term miltefosine-allopurinol treatment.^244,250,251 The effect of treatment on parasite transmission to sand flies or
on Leishmania-specific cell-mediated immunity was not evaluated in
either RCT or in any other study. There was a significant reduction in IFA
titers starting at 28 days of treatment and continuing over the next 6 months.²⁴⁰

²⁴⁰ or 12.5-16.7%²⁴⁹ of the dogs, they were not severe, and included
vomiting, soft stools and diarrhea.²⁴⁹ Induction of miltefosine-resistant strains of L.
infantum was confirmed in a single dog.²¹⁸

Conclusion: Miltefosine-allopurinol
combination is indicated for the treatment of CanL due to the generally
consistent (apart from the evolution of parasitic load), albeit of limited
quality, LoE showing that it results in clinical improvement, in amelioration
of clinically important clinicopathologic abnormalities, in downregulation of
humoral immunity and that it is safe and non-nephrotoxic (SORT: moderate). To
increase the SORT, additional RCTs are necessary and some features of this
treatment (evolution of infectiousness to sand flies and of cell-mediated
immunity) should be studied. Miltefosine daily recommended dose is 2 mg/kg for
4 weeks, because the modified dosage regimen (1.2 mg/kg for the first 5 days
followed by 2.5 mg/kg for 25 days), despite the higher cumulative dose of the
drug that is administered, does not offer any appreciable clinical benefit and
its safety has not been evaluated in toxicologic studies (SORT: weak). Repeated
administration of miltefosine, like unnecessary extended allopurinol administration
periods, should be avoided due to the risk of induction of resistant strains of
L. infantum (SORT: moderate).

8.3.10.6. Allopurinol-aminosidine combination

            The
efficacy and safety of allopurinol-aminosidine combination have been evaluated
in two RCTs (Table S17),^182,239 of 2²³⁹ or 6¹⁸² month-duration, and including 20 dogs with CanL.
Allopurinol was administered at the usual dose (10 mg/kg twice daily for 2 or 6
months) and aminosidine at the recommended dose of 15 mg/kg SC once daily for
28 days. The diagnosis of CanL was confirmed by serology and lymph node and/or
bone marrow microscopy, and CKD IRIS stage III or IV was an exclusion
criterion. In both studies, the comparator was the meglumine
antimoniate-allopurinol combination, and their quality is intermediate²³⁹ or high.¹⁸²

            One
dog died of unknown reasons during the first week of treatment. Of the
remaining dogs, 21.4% (4/19) were clinically cured at 6 months, using a very
stringent clinical scoring system, and there was a significant reduction of the
number of clinical signs per dog, in the prevalence of 8/16 different clinical
signs that were present on day 0, and in the severity of 12/16 clinical signs.
The prevalence and/or severity of most clinicopathologic abnormalities (anemia,
hyperproteinemia, hyperglobulinemia, low albumin-globulin ratio, CRP) was
significantly lower at 6 months compared to time 0, and there was no evidence
of nephrotoxicity based on BUN, creatinine, inorganic phosphorus, and UPC at 2
and 6 months.

Lymph node and bone marrow microscopy
showed a reduction of parasitic load, starting at 4 weeks. Bone marrow
parasitic load, based on qPCR, was significantly lower at 6 months than on day
0; moreover, at 6 months, 26.3% (5/19) dogs were negative on bone marrow qPCR
and microscopy and lymph node microscopy. No data are available on the
infectivity of treated dogs to sand flies.

Allopurinol-aminosidine combination
treatment induced Leishmania-specific cell-mediated immunity,
exemplified by the higher number of dogs with positive leishmanin skin test at
6 months (47.4%) compared to baseline (10.5%). In parallel, IFA titers were
significantly lower from the first month, and at the end of the trial 21.1%
(4/19) dogs were seronegative.

In addition to the single dog that died
suddenly, aminosidine injection site reactions were common (55%).¹⁸² However, there was no evidence of nephrotoxicity and ototoxicity
was excluded based on brainstem auditory evoked responses and normal
neurological examination.²³⁹

            A
direct comparison between allopurinol-aminosidine and meglumine
antimoniate-allopurinol combination showed some advantages of the latter, such
as increased prevalence of dogs with no clinical signs, lower number of
clinical signs and clinicopathologic abnormalities per dog, and higher chance
for reduction of hyperglobulinemia and IFA titers at 6 months.¹⁸² Also, being an aminoglycoside,
aminosidine may promote bacterial resistance.

Conclusion: Allopurinol-aminosidine
is indicated for the treatment of CanL due to the good quality LoE showing that
it results in clinical improvement or cure, amelioration of clinically
important clinicopathologic abnormalities, reduction of parasitic load,
upregulation of parasite-specific cell-mediated immunity with downregulation of
humoral immunity. The combination is reasonably safe and non-nephrotoxic or
ototoxic (SORT: strong). Allopurinol dose should be 10 mg/kg orally twice daily for at least
6 months, and aminosidine should be administered at 15 mg/kg SC once daily for
28 days (SORT:
weak). The case of sudden death during aminosidine administration indicates
that close monitoring is necessary (SORT: strong). Since meglumine
antimoniate-allopurinol combination seems to be more effective and aminosidine
may promote bacterial resistance, allopurinol-aminosidine should be considered
a second-line treatment of CanL (SORT: strong) that may be particularly
valuable in dogs that relapse despite multiple courses of meglumine antimoniate
and/or miltefosine and may be at increased risk to harbor resistant parasites
(SORT: weak).

8.4. Immunomodulators

8.4.1. Domperidone

            Domperidone
is a prokinetic and antiemetic drug acting through antagonism of dopamine D2
receptors. By the same mechanism, in the central nervous system domperidone
induces serotonin and, subsequently, prolactin release. The latter stimulates
innate (e.g., increased neutrophil oxidative activity) and cell-mediated (e.g.,
increased Leishmania antigen-stimulated INF-γ production by PBMCs)
immunity.²⁵² Onset of efficacy is quite fast (from the 2^nd
day of administration), and the registered dose is 0.5 mg/kg orally once daily
for 1 month, that is repeated in cycles separated by a 3-month off-drug period.

            The
efficacy and safety of domperidone for the treatment of CanL have been
evaluated in one RCT (Table S18).²⁵³ In that study, 30 seropositive dogs without clinical
signs of CanL but with CKD IRIS stage I or II (that was assumed to be due to
CanL) were fed a renal diet for 11 months with (treatment group) or without
(control group) domperidone. In the treatment group, domperidone was
administered twice, starting on day 90 and on day 210. The quality of the study
is intermediate.

            No
information about the evolution of clinical signs is provided, but 1/15 (6.7%)
dogs from each group died of CanL. In the domperidone-treated dogs, serum
creatinine and SDMA concentrations did not differ between the beginning and the
end of the study, whereas both biochemical markers deteriorated significantly
in the control group. No treatment-related adverse effects were recorded.

            In an
open trial, domperidone did not prevent the appearance of new clinical signs
and/or clinicopathologic abnormalities of CanL in 25% (3/12) seropositive dogs
with CKD. The remaining 9/12 dogs remained stable and, by the end of the
6-month study period, they had significantly decreased globulins,
gamma-globulins, and CRP compared to time 0, no change in their UPC, and
decreased ELISA ODs.²⁵⁴ In another open trial, 7.1% (2/28) dogs died of CanL,
but 85.7% (24/28) showed clinical improvement, associated with increased
diameter of leishmanin skin test reaction and increased PBMC proliferation in
response to parasite antigen.²⁵⁵ The impact of domperidone administration on the
infectivity of treated dogs to sand flies has not been studied.

Conclusion: Domperidone
is not indicated for the treatment of CanL because it has been tested only in a
few dogs from a specific subgroup that presented early-stage CKD, without additional
clinical signs or clinicopathologic abnormalities of the disease, making
impossible to determine the overall efficacy of the treatment (SORT: weak).

8.4.2. Nutritional supplements

            A
nutritional supplement with antioxidant properties, of mixed marine and plant
origin, marketed under the trade name DiLsh^TM
was tested in one RCT, where it was compared with meglumine
antimoniate-allopurinol combination.²³⁷ In this study, the nutritional supplement was
administered at a daily dose of 0.5 g/kg orally (in the food) for 3 months to
15 dogs with CanL of unclear severity, that was confirmed by serology and bone
marrow PCR. The quality of the study is intermediate.

            By the
end of the trial, there was significant reduction of total clinical score and
none of the dogs had died of CanL. Of the clinicopathologic abnormalities,
there was only a significant decrease of gamma-globulins and no significant
changes of total protein, albumin, BUN, and creatinine concentrations. The
evolution of parasitic load and infectivity to sand flies was not examined,
cell-mediated immunity was evaluated with a non-validated approach (measurement
of IL-6, IL-10, TNF-a, and leptin concentrations in serum) and there was a
significant reduction of ELISA ODs. Adverse effects were not recorded.

Conclusion: The
nutritional supplement DiLsh^TM is not indicated for the treatment of
CanL because of the limited information on critical features of this treatment
such as the evolution of parasitic load, infectivity to sand flies and Leishmania-specific
cell-mediated immunity.

8.4.3. Monoclonal antibody against canine IL-10 receptor

            In a
single RCT (Table S20) of 6-month duration and intermediate quality,
anti-canine IL-10 receptor monoclonal antibodies were compared to liposomal
meglumine antimoniate.²⁰⁷ Two IM injections (4 mg/kg) of the monoclonal antibody
were administered, 21-days apart, to 11 dogs with CanL of unknown severity,
confirmed by bone marrow culture and serology.

            There
was an initial (days 30 and 90) non-significant improvement, followed by
deterioration of total clinical score that, at the end of the trial, was almost
the same as on day 0. No changes were found in hematocrit, platelet count,
total protein, globulin, BUN, and creatinine concentrations or in bone marrow
parasitic density, whereas infectivity to sand flies and humoral immunity were
not tested. At different time points there were multiple, often temporary,
changes of PBMCs immunophenotype. On day 180 there was a significant decrease
of T cell proliferation in response to Leishmania antigen, and of CD5-
CD16+ natural killer cell absolute numbers, compared to day 0. No adverse
effects were observed.

Conclusion: Monoclonal
antibodies against canine IL-10 receptor are not indicated for the treatment of
CanL due to lack of efficacy (SORT: moderate).

8.4.4. Vaccines

            A
vaccine containing a partially purified fraction derived from L. infantum
promastigotes with a molecular weight of 67-94 KDa, called LiF2, was
administered to eight dogs with CanL in one RCT (Table S21) of intermediate
quality.¹⁹⁰ The dose of the vaccine was 50 μg/dog administered
intramuscularly (IM) for 3 times at weekly intervals. The diagnosis of CanL was
confirmed by bone marrow microscopy but the severity of the disease was
unclear. In this study, the vaccine was compared to meglumine antimoniate and
to the meglumine antimoniate-LiF2 vaccine combination. After 3 months, 25%
(2/8) of the dogs were clinically cured and 75% (6/8) improved and at 6 months
the cure rate was 100% (8/8). At both time points, bone marrow parasitic density
decreased, 25% (2/8) dogs were negative on both microscopy and culture and the
parasiticidal activity of macrophages increased (at least in some dogs). No
information on the evolution of clinicopathologic abnormalities, infectivity to
sand flies, humoral immunity, and adverse effects was provided.

Conclusion: The LiF2
vaccine is not indicated for the treatment of CanL because of the limited
information on critical features of this treatment such as the evolution of
clinicopathologic abnormalities, infectivity to sand flies, Leishmania-specific
humoral immunity and adverse effects.

In one RCT (Table
S21), the vaccine Leish-110f^®, containing a
polyprotein composed of three recombinant Leishmania proteins (TSA,
LmSI1, and LeIF) at 25 μg/dose, and the adjuvant monophosphoryl lipid A (MPL)
plus squalene in a stable emulsion (MPL-SE^®) at 25 μg/dose was
administered SC three times at 3-week intervals, to six dogs with CanL. All
dogs were considered “symptomatic” (i.e., not “oligosymptomatic”), the
diagnosis of CanL was confirmed by microscopy and/or culture, the duration of
the RCT was 6 months, and the efficacy of the vaccine was compared to meglumine
antimoniate, meglumine antimoniate-vaccine combination, MPL-SE^®
adjuvant, and placebo.¹⁸⁹ At the end of the study, clinical improvement
was seen in none of the dogs, the evolution of their major clinicopathologic
abnormalities is not clearly reported and all dogs were still positive on bone
marrow and/or skin microscopy and/or culture. There was no evidence of
stimulation of cell-mediated immunity based on PBMC proliferation in response
to parasite antigen, the anti-Leishmania IgG titer was higher compared
to day 0, and none of the dogs became seronegative. No adverse effects were reported.¹⁸⁹

            In
another publication describing the results of two RCTs (Table S21) of
intermediate quality,¹⁹¹ the same vaccine with the same adjuvant, now
called Leish-111f^®, was administered at a lower dose (20 μg vaccine
plus 20 μg adjuvant), SC four or six times at 1-week intervals to 18 (study #1)
or 10 (study #2) dogs with CanL of unknown severity, confirmed by microscopy, culture
or serology. The vaccine was compared to meglumine antimoniate, meglumine
antimoniate-vaccine combination, the adjuvant, and placebo (saline) or no
treatment. In the first study, clinical improvement was seen in all 18 dogs 6
months after the first vaccination, but 2.5 years later, of the 12 dogs that
had not been lost to follow-up or died for unrelated reasons, 75% (9/12) were
considered clinically healthy, 25% (3/12) had a relapse, and two of them had
died of CanL. In the second RCT, the clinical efficacy was much lower. At 6
months 50% (5/10 dogs) had improved, whereas the remaining 5/10 dogs
deteriorated between 1-4 months and they either received rescue treatment or
died. Interestingly, the responders were mainly dogs with low severity of CanL on
day 0. No additional information is provided, except that in study #2 6/10
(60%) dogs were negative on microscopy or culture at 6 months.¹⁹¹

Conclusion: The
vaccine Leish-110f^® with the adjuvant MPL-SE^® is not
indicated for the treatment of CanL due to low efficacy in two out of three
published RCTs (SORT: moderate).

            The
efficacy and safety of a vaccine containing recombinant cysteine proteinase of Leishmania
(rLdccys1) as antigen and Propionibacterium acnes as adjuvant were
tested in one RCT (Table S21).²⁵⁶ The vaccine was administered at a dose of 500 μg
rLdccys1 plus 500 μg adjuvant SC three times at 1-month intervals to 10 dogs
with CanL of undetermined severity (without pancytopenia or creatinine
concentration >2 mg/dl) confirmed by bone marrow culture and serology. The
control groups received only the adjuvant or placebo, and the quality of the
study is intermediate. The clinical score of vaccinated dogs did not improve
after 1, 2, 3 or 4 months, and they died after 12-14 months. Vaccine
antigen-specific cell-mediated and humoral response was apparent during
treatment, and, by the time of death, their spleen parasitic burden was 7-log
lower compared to the controls. Local adverse effects of transient nature were
recorded in most dogs.

Conclusion: The
vaccine containing rLdccys1 antigen with the adjuvant P. acnes is not
indicated for the treatment of CanL due to lack of efficacy (SORT: moderate).

            A
vaccine containing L. braziliensis antigen and the adjuvant MPL was tested in one RCT (Table
S21) of 90-day duration.²⁵⁷ The vaccine was administered
SC once daily to 10 dogs, at gradually increased doses for the first 5 days and
then at the maximal dose (600 μg vaccine plus 25 μg adjuvant) until day 10,
followed by a 10-day discontinuation period. This was followed by 10-day
re-administration at the maximal dose, a 10-day discontinuation and a 10-day
re-administration. The control dogs received adjuvant or no treatment, the
diagnosis of CanL was confirmed by serology and PCR, the severity of CanL was unknown,
and the quality of the study is intermediate. Dogs were euthanized after 3
months and the vaccinated group was found to have 96% lower spleen parasitic
burden, based on qPCR, compared to controls. The expression of IL-12, INF-γ,
TNF-a and inducible nitric oxide synthetase (iNOS) mRNA in the spleen was
significantly higher, and that of IL-10 and transforming growth factor-b1
(TGF-b1) was significantly lower compared to controls. No clinical information
is provided in the RCT. However, in a non-randomized adjuvant-controlled trial
of 5-month duration, there was clinical improvement in 70% of vaccinated dogs,
which was accompanied by normalization of red blood cell parameters, platelet
count, BUN, and creatinine, reduction of bone marrow parasitic load and
infectivity to sand flies, activation of Leishmania-specific
cell-mediated immunity, increased transcription of INF-γ and TNF-a, and
decreased transcription of IL-4 and IL-10.²⁵⁸ .

Conclusion: The
vaccine containing L. braziliensis antigen with the adjuvant MPL is not
indicated for the treatment of CanL due to the limited information on most
critical features of this treatment such as the evolution of clinical signs and
clinicopathologic abnormalities, infectivity to sand flies and Leishmania-specific
humoral immunity, and the relatively low clinical response rate in a
non-randomized trial (SORT: moderate).

8.5. Combinations of drugs with direct
anti-Leishmania activity and immunomodulators

8.5.1. Meglumine antimoniate-nutritional supplement
combination

            A
nutritional supplement containing nucleotides and an AHCC compound (Impromune^®)
may modulate immune responses through non-specific stimulation of cell-mediated
immunity and Th1 cytokine production.^241,259 The efficacy and safety of the combination of this
supplement with meglumine antimoniate were tested in a single RCT (Table S22).²⁴¹ In this study, 32 dogs with CanL were treated with
meglumine antimoniate at the recommended dose (100 mg/kg SC daily) for 4 weeks,
and with the dietary supplement at 32 mg/kg of nucleotides plus 17 mg/kg AHCC
daily for 6 months. These dogs were compared to 38 dogs with CanL treated with
the standard meglumine antimoniate-allopurinol combination. The confirmation of
CanL diagnosis was based on positive serology and bone marrow or lymph node
microscopy and/or PCR. The severity of the clinical picture of CanL, evaluated
by a clinical scoring system with a maximum value of 55, was probably mild
(clinical score 7.67 ± 3.84). The quality of the study is intermediate.²⁴¹

            The
number of dogs achieving complete or partial clinical cure by the end of the
study is not reported, but there was a significant reduction in the clinical
score, that probably was first witnessed at the end of meglumine antimoniate
administration. At 6 months, none of the dogs had died of CanL and the clinical
score was significantly lower compared to dogs treated with meglumine
antimoniate-allopurinol combination. There was a significant decrease of total
protein, gamma-globulin, CRP, and ferritin concentrations, a significant
increase of albumin on days 30 and 180 compared to baseline, and there were no
changes in the biomarkers of kidney function (BUN, creatinine, UPC).

            At the
end of the study, the parasitic load (bone marrow or lymph node qPCR) was
reduced compared to time 0; the effect of treatment on the infectivity to sand
flies was not tested.

            The
effect of treatment on Leishmania-specific cell-mediated immunity was
examined using a non-validated approach (measurement of CD4+ and CD8+ cells in
unstimulated blood samples) and a significant reduction of ELISA ODs at the end
of the study compared to baseline was demonstrated.

            No
treatment-related adverse effects were reported.²⁴¹

Conclusion: Despite
the improvement of clinical signs and clinicopathologic abnormalities and the
reduced parasitic load and humoral response (SORT: moderate), the meglumine
antimoniate-dietary nucleotide plus active hexose correlated compound
combination cannot be recommended as a routine alternative to the standard
meglumine antimoniate-allopurinol combination because the efficacy was tested
mainly in dogs with CanL of mild severity (SORT: weak) and due to lack of
information on some critical features of this treatment (percentage of dogs
achieving clinical cure or improvement, percentage of dogs with amelioration of
clinicopathologic abnormalities, infectivity to sand flies, effect on Leishmania-specific
cell-mediated immunity).

8.5.2. Meglumine antimoniate-vaccines combination

            The
combination of meglumine antimoniate at a high dose (300 mg/kg IM every other day
for 20 administrations) with the LiF2 vaccine (50 μg/dog IM 3 times at weekly
intervals) was compared to meglumine antimoniate monotherapy and to LiF2
monotherapy in one RCT (Table S23), that included eight dogs in each treatment
group.¹⁹⁰ The diagnosis of CanL was confirmed by microscopy of
bone marrow aspirates, the severity of CanL is unclear, and the quality of the
study is intermediate.¹⁹⁰

            At 3
months all dogs were considered clinically cured but no information on the
evolution of clinicopathologic abnormalities is available. At 3- and 6-months
bone marrow aspirates by microscopy and culture were negative in 8/8 (100%) of
the dogs but their infectivity to sand flies was not studied. The
leishmanicidal activity of macrophages increased after treatment but there is
no information about Leishmania-specific humoral immune response. No adverse
effects were encountered.¹⁹⁰

Conclusion: The
meglumine antimoniate-LiF2 vaccine combination is not indicated for the
treatment of CanL because of the limited information on critical features of
this treatment such as the evolution of clinicopathologic abnormalities,
infectivity to sand flies and Leishmania-specific humoral immunity.

            The
safety and efficacy of meglumine antimoniate and vaccine Leish-110f^®/
Leish-111f^® combination were examined in two RCTs (Table S23).^189,191 The number of treated dogs was six¹⁸⁹ and 13,¹⁹¹ the dosage regimen of meglumine antimoniate varied
(100 mg/kg IM, daily for 10 days followed by 10-day discontinuation and then by
administration for 10 more days¹⁸⁹ or 20 mg/kg IV daily for 30 days)¹⁹¹ and the vaccine was administered at 20 μg (plus 20 or
25 μg of MPL-SE^®) per dog SC once weekly for 4 times¹⁹¹ or every 3 weeks for 3 times.¹⁸⁹ The combination treatment was compared to meglumine
antimoniate^189,191 and vaccine^189,191 monotherapies, to vaccine adjuvant,¹⁸⁹ and to placebo¹⁸⁹ or no treatment.¹⁹¹ The diagnosis of CanL was confirmed by microscopy
(bone marrow, lymph node, spleen, skin),^189,191 culture (bone marrow, spleen)^189,191 and/or serology,¹⁹¹ and the severity of CanL is not reported. The quality
of these studies is low¹⁸⁹ or intermediate.¹⁹¹

            At 6
months after treatment initiation 0-16.7% dogs had died, 50% were clinically
cured and 33.3-92.3% improved. Evolution of clinicopathologic abnormalities is
reported in one of the studies,¹⁸⁹ where there was a significant increase in hematocrit
and albumin concentration after 1-2 months and normalization of gamma-globulins
after 6 months, accompanied by negative bone marrow microscopy and culture,
negative skin microscopy, and negative xenodiagnosis in 40% (2/5) of the
surviving dogs. The effect of treatment on Leishmania-specific cell-mediated
immunity was not different from placebo, but there was a decline in IgG
responses after 6 months with 40% (2/5) dogs becoming seronegative.¹⁸⁹ No adverse effects were reported.

Conclusion: The
meglumine antimoniate- Leish-110f^®/ Leish-111f^® vaccine
combination is not indicated for the treatment of CanL because of the lack of
an obvious benefit compared to meglumine antimoniate monotherapy (SORT:
moderate).

8.5.3. Meglumine antimoniate-allopurinol-domperidone
combination

            The
efficacy and safety of the addition of domperidone to meglumine
antimoniate-allopurinol combination treatment was evaluated in a single RCT (Table
S24) of very short (3 weeks) duration.²⁴³ During the study, 36 dogs with CanL stage C (CLWG
classification system) were treated with meglumine antimoniate and allopurinol
at the recommended doses (100 mg/kg SC once daily and 10 mg/kg orally twice
daily, respectively), whereas half of them (18/36) were randomly assigned to
also receive domperidone at the registered dose (0.5 mg/kg orally once daily).
The diagnosis of CanL was confirmed by skin, lymph node, and/or bone marrow
microscopy and/or PCR and by positive serology. The quality of the study is
intermediate.

            Clinical
signs of CanL improved in all dogs by the end of the 2^nd week of
treatment (however, clinical improvement by that time was an inclusion
criterion) and none of them died or was euthanized. The only information on the
evolution of clinicopathologic abnormalities is that serum electrophoresis
profile was not restored by the end of the study, and that CRP, after a
transient increase on day 3, gradually decreased, being significantly lower on
days 14 and 21 compared to baseline. No information on the evolution of
parasitic density, infectivity to sand flies, parasite-specific cell-mediated
or humoral immunity was provided, and no adverse effects were reported. There
were no important difference between the two groups.²⁴³

Conclusion: The
addition of domperidone to the meglumine antimoniate-allopurinol combination
treatment cannot be recommended for the treatment of CanL because of the lack
of an obvious benefit (SORT: moderate) and the limited information on some
critical features of this treatment such as the evolution of parasitic density,
infectivity to sand flies and Leishmania-specific cell-mediated and
humoral immunity.

8.5.4. Meglumine antimoniate-allopurinol-deslorelin
combination

            Deslorelin
is a gonadotropin-releasing hormone agonist that is used for chemical
sterilization of male and female dogs and cats.²⁶⁰ Under the assumption that long-term blockage of
testosterone production in intact male dogs may have a beneficial effect on the
immune response against the parasite, the efficacy and safety of the addition
of deslorelin to meglumine antimoniate-allopurinol combination were tested in a
single RCT (Table S25).²⁴² In this study, meglumine antimoniate and allopurinol
were administered at the recommended therapeutic regimen (100 mg/kg SC daily
for 4 weeks, and 10 mg/kg orally twice daily for 6 months, respectively) and a
single 4.7 mg deslorelin implant was injected SC. Eleven intact male dogs
received deslorelin (and 12 intact male dogs were treated only with meglumine
antimoniate and allopurinol), CanL diagnosis was confirmed by positive serology
and positive microscopy or PCR of bone marrow and/or lymph nodes, and all dogs
were at CanL LeishVet stages IIa, IIb, or III. The quality of the study is
intermediate.²⁴² 

            Clinical
score at 3 and 6 months was significantly lower than on day 0 and compared to
the control (meglumine antimoniate-allopurinol) group. Similarly, IFA titers
were significantly lower at 3 and 6 months than on day 0, and significantly
lower than the control group at 6 months. No treatment-related adverse effects
were recorded although 1/11 (9.1%) dog died due to a seemingly unrelated cause
(congestive heart failure).²⁴²

            There
are no data on relapses after treatment discontinuation, the evolution of
clinically important clinicopathologic abnormalities, parasitic load, and
infectivity to sand flies, or possible effects to Leishmania-specific
cell-mediated immune response.

Conclusion: In
intact male dogs, the addition of deslorelin to meglumine
antimoniate-allopurinol treatment may offer some clinical benefits and seems to
be safe (SORT: moderate). However, more information on critical features of
this therapeutic strategy (evolution of clinicopathologic abnormalities,
parasitic load, infectivity to sand flies and Leishmania-specific
cell-mediated immunity) is needed to be considered as standard-of-care (SORT:
weak).

8.5.5. Allopurinol-metronidazole-ketoconazole-n-3 fatty
acid-B vitamin combination

            In
addition to allopurinol and metronidazole, ketoconazole has direct anti-Leishmania
activity, due to the inhibition of parasite cytochrome P450 enzymes, leading to
accumulation of 14-methyl sterols that may affect cell membrane fluidity and
permeability.²¹⁹ Ketoconazole has been tested as monotherapy for CanL
in an open trial including 14 dogs with promising results.²⁶¹ N-3 fatty acids were hypothesized to be helpful for
the treatment of CanL due to their anti-inflammatory and antioxidant
properties, whereas the role, if any, of B-complex vitamins, is obscure.²⁶² The efficacy and safety of
allopurinol-metronidazole-ketoconazole-n-3 fatty acid-B vitamins combination
were tested on one RCT (Table S26).²⁶² In that study allopurinol was administered at the
recommended dose (10 mg/kg twice daily) for either 270 or 320 days,
metronidazole at 25 mg/kg twice daily for 30 days, ketoconazole at 10 mg/kg
once daily for 40 days, the n-3 (eicosapentaenoic and docosahexaenoic) fatty
acids at 1,000 mg/kg once daily for 300 or 360 days, and the B complex vitamin
liquid formulation at 2 drops/kg once daily for 300 or 360 days. All 30 dogs
were treated will all the above but in a different order: one group (group A;
n=15) was treated during the first 30 days with n-3 fatty acids and B vitamins
only, then metronidazole (for 30 days) and ketoconazole (for 40 days) were
added and were replaced by allopurinol from day 90 until day 360. In the second
group (group B; n=15) metronidazole and ketoconazole were the only
interventions during the first 30 and 40 days, respectively, allopurinol was
started on day 41 and continued until day 360, and the n-3 fatty acids and B
vitamins started on day 60 and continued until day 360. The diagnosis of CanL
was confirmed by bone marrow and/or lymph node microscopy and PCR and all dogs
were classified as LeishVet stage I or II. The quality of the study is low.²⁶²

            Irrespectively
of the treatment group, on day 360 all dogs that were not lost to follow up
(6/15 and 12/15 for groups A and B, respectively), presented at least one
clinical sign of CanL, but their clinical scores improved, starting at 3 (group
A) or 6-12 (group B) months. At the end of the study, 20-27.3% of them were
classified at a lower stage of CanL (LeishVet stage I) compared to day 0
(LeishVet stage II). At the same time point, and considering only those dogs
that presented each of the following clinicopathologic abnormalities at
baseline, hematocrit normalized in 50% (1/2) group A and in 57.1% (4/7) group B
dogs, platelet count normalized in 66.7% (2/3) group B dogs, total protein
concentration normalized in 25% (1/4) group A and in 10% (1/10) group B dogs,
albumin concentration normalized in 0% (0/1) group A and in 71.4% (5/7) group B
dogs, globulin concentration normalized in 25% (1/4) group A and in 30% (3/10)
group B dogs, and albumin/globulin ratio normalized in 66.7% (2/3) group A and
in 14.3% (1/7) group B dogs. BUN concentration normalized in 50% (1/2) group A
and in 100% (3/3) group B dogs, creatinine concentration normalized in one
group A and one group B dog, and UPC normalized in one group A and in 60% (3/5)
group B dogs. In addition, some dogs showed improvement without normalization
of their hematocrit (1/2 in group A), platelet count (1/3 in group B), total protein
(1/4 in group A and 5/10 in group B), albumin (1/1 in group A and 1/7 in group
B) and globulin (2/4 in group A and 3/10 in group B) concentrations,
albumin/globulin ratio (3/7 in group B), BUN concentration (1/2 in group A) and
UPC (1/5 in group B). The improvement of clinicopathologic abnormalities was
evident between 60 and 360 days but was not accompanied by a reduction of bone
marrow parasitic load based on qPCR or of ELISA ODs, that did not differ
between baseline and the end of the study in either group; at the later time
point, 20% (1/5) and 18.2% (2/11) group A and group B dogs, respectively, were
bone marrow qPCR negative. The evolution of infectivity to sand flies and
cell-mediated immunity were not examined and no adverse effects were recorded.²⁶²

Conclusion: Allopurinol-metronidazole-ketoconazole-n-3
fatty acid-B vitamin combination cannot be recommended for the treatment of
CanL due to the moderate efficacy (SORT: moderate) and the lack of information
on critical features of this treatment such as the evolution of infectivity to
sand flies and Leishmania-specific cell-mediated immunity. Due to the
design of the study, it is not possible to draw conclusions about the efficacy
of ketoconazole. Due to lack of a standardized diet, it is unclear if the
addition of n-3 fatty acids and B complex vitamins offers some benefit (SORT: weak).
Also, it is questionable if it is prudent to delay the administration of anti-Leishmania
drugs by 1 month during which only n-3 fatty acids and B complex vitamins are
administered (SORT: weak). However, n-3 fatty acids may have some beneficial
effects on proteinuria (SORT: weak).

8.5.6. Allopurinol-vaccine combination

            The
efficacy and safety of the combination of allopurinol and LeishF2 vaccine was
tested in a single RCT (Table S27).²²² Allopurinol was administered for 3 months at the
recommended daily dose (20 mg/kg) and the vaccine was injected 6 times at
3-week intervals. The combination was administered to 8 dogs and was compared
to allopurinol monotherapy and no treatment. The diagnosis of CanL was
confirmed by bone marrow qPCR, but the severity of the disease is not reported.
The quality of the study is intermediate.²²²

            After
1 year (9 months after treatment discontinuation), none of the dogs had died of
CanL, and their total clinical score was significantly lower than the no
treatment group, but not compared to allopurinol monotherapy. No further
information about the clinical signs (e.g. percentage of dogs with clinical
cure or improvement) and about the evolution of clinicopathologic parameters is
provided. Bone marrow qPCR was negative in 7/8 dogs at 2 months and in all dogs
at 6 and 12 months. Moreover, at the latter time point, liver and kidney qPCR
was negative in all dogs and lymph node and spleen PCR was positive in 1/8
dogs. At 2-, 6- and 12-months bone marrow parasitic density was significantly
lower compared to the no treatment group, at 13 months it was also lower
compared to allopurinol monotherapy, and there was no tendency for increased
parasitic density between 3 (treatment end) and 12 months (end of the study).
No information on infectivity to sand flies, parasite-specific cell-mediated or
humoral immunity were provided, and no adverse effects were reported.²²²

Conclusion: The
allopurinol-LeishF2 vaccine combination treatment is not indicated for the
treatment of CanL because of the lack of an obvious benefit over allopurinol
monotherapy, except for the lower parasitic density after 1 year (SORT:
moderate), and due to the limited information on some critical features of this
treatment such as the evolution of clinicopathologic abnormalities, infectivity
to sand flies and Leishmania-specific cell-mediated and humoral immunity.

8.6. Symptomatic treatment

            Some
manifestations of CanL may need additional therapeutic interventions. The most
common problems are proteinuria, arterial hypertension, uremic syndrome, and
rhinorrhagia. Unfortunately, there is a shortage of clinical trials on the
optimal management of these manifestations in the setting of CanL.

            Proteinuria
may have multiple negative consequences, including deterioration of kidney
excretory function and CKD, arterial hypertension, hypoalbuminemia, nephrotic
syndrome, and pulmonary thromboembolism.^233,245,263 Since recommended treatments for CanL can decrease or ameliorate
proteinuria,^239-241 and depending on UPC values, creatinine and iP
concentrations, a waiting period, varying between 3 days to 8 weeks, before
adding symptomatic treatment for proteinuria has been proposed.^172,264 Possible interventions include a diet low in
phosphorous, angiotensin converting enzyme inhibitors (ACEIs, like benazepril
or enalapril), angiotensin receptor blockers (e.g., telmisartan), aldosterone
receptor blockers (e.g., spironolactone), n-3 fatty acid supplements, and, as a
last resort, immunosuppressive and/or cytotoxic drugs like prednisolone,
mycophenolate mofetil, cyclophosphamide, chlorambucil, ciclosporin and/or
azathioprine.^172,265 Usually ACEIs are the first line of medical treatment
for proteinuria, and in an open clinical trial on 12 dogs with CanL at LeishVet
stage II or III with CKD IRIS stage I or II, feeding a kidney diet (12/12 dogs)
and administration of benazepril (6/12 dogs) along with meglumine
antimoniate-allopurinol combination resulted in significant reduction of UPC at
3 months, although proteinuria was still present in 3/5 initially proteinuric
dogs.²⁴⁵ Caution is advised if immunosuppressive and/or cytotoxic
drugs need to be used because they may lead to initial treatment failure or
relapse.^266,267 The decision to start immunosuppressive treatment necessitates
confirmation of immune-mediated glomerulonephritis through histopathology
and/or direct immunofluorescence and/or electron microscopy.¹⁷² Also, concurrent administration of azathioprine and
allopurinol is contraindicated due to the risk of myelosuppression.⁹⁴

Conclusion: The
administration of benazepril in dogs with CanL and proteinuria may be
beneficial (SORT: weak). In unresponsive cases, additional therapeutic
interventions may be considered, but the benefit/harm ratio should be addressed
on a case-by-case basis, especially if immunosuppressive and/or cytotoxic
medication is prescribed (SORT: weak).         

            Hypertension
is very common in dogs with CanL,²³³ but there are no reports on the efficacy of
antihypertensive treatment. Therefore, the typical therapeutic strategy for the
symptomatic management of canine hypertension can be followed, starting with
ACEIs and, if they are not effective enough, adding angiotensin receptor
blockers and then calcium channel blockers (e.g., amlodipine).^172,265,268

            Apart
from the case report of a dog with acute kidney injury that responded to
hemodialysis and later to standard treatment against CanL (initially
allopurinol and later addition of miltefosine),⁵⁹ there are no data on treatment of advanced CKD/uremic
syndrome in this disease. Due to the poor prognosis, euthanasia may be
considered. Otherwise, standard symptomatic treatment should start as soon as
possible, and, depending on the case, it may include hemodialysis,
fluid/electrolyte parenteral administration, kidney diet, phosphate binders
(e.g., aluminum hydroxide, calcium acetate or carbonate), calcitriol,
antiemetics (e.g., maropitant, ondansetron), stimulators of erythropoiesis
(e.g., darbepoetin-a), etc.^268,269 After stabilization, treatment for CanL should start
but there are scarce data on the safety of recommended drugs in these patients,
except for allopurinol and marbofloxacin.^232,270 Due to the low efficacy of allopurinol monotherapy and the relapses after
marbofloxacin discontinuation, combination treatment should be considered after
a certain period decided on a case-by-case basis. The potential of the
aminoglycoside aminosidine to cause additional kidney damage and some evidence
of histologic changes in the kidneys of healthy dogs after meglumine
antimoniate but not after miltefosine administration²⁷¹ may imply that the miltefosine-allopurinol combination
is preferable.

Conclusion: Dogs
with CanL and uremic syndrome should first be stabilized with the symptomatic
treatment for CKD, then allopurinol or marbofloxacin can be administered, and
finally miltefosine-allopurinol combination may be the preferred treatment to
control the disease (SORT: weak).

            Massive
bleeding from the nose can cause blood-loss anemia and even death. In the
absence of studies, symptomatic treatment should be adjusted to the severity of
bleeding and may include cold packs, nasal cavity tamponade, temporal ligation
of carotid artery, blood transfusion, and oxygen supplementation. Since
ulcerative rhinitis and thrombocytopathy are major causes of nasal bleeding in
CanL,⁶⁶ a short course of glucocorticoids at anti-inflammatory
dose (e.g., prednisolone or prednisone, 0.5-1 mg/kg orally once daily for 7-21
days) may be beneficial by reducing nasal inflammation and restoring platelet
function.²⁷²

Conclusion: Rhinorrhagia
in dogs with CanL should be treated symptomatically and perhaps with a short
course of glucocorticoids at anti-inflammatory dose (SORT: weak).

8.7. Treatment of concurrent diseases

            A wide
variety of comorbidities, mainly of hormonal, neoplastic, infectious and
parasitic etiology, have been reported in dogs with CanL, and have been
attributed to dual breed predisposition, co-endemicity, inadequate protection
from insect/vector bites, CanL-induced immunosuppression and/or the effect of
comorbidities to the immune system that may render a resistant, subclinically
infected dog to develop CanL.^38,273-275 It has been proposed that comorbidities should be actively searched in
dogs with CanL, especially those with atypical manifestations and poor response
to treatment. Their simultaneous (or sequential) treatment may improve the
final outcome.^276-278

Conclusion: Treatment
of comorbidities may increase the efficacy of treatment for CanL (SORT: weak).

            An uncommon comorbidity involving the skin is a pustular dermatitis,
different from the pustular form of CanL, resembling pemphigus foliaceus
clinically, cytologically, histopathologically,⁹⁴ and immunologically (S. Colombo, P. Bizikova: personal
communication 2024). Reportedly,
these lesions do not regress during anti-Leishmania treatment and
necessitate administration of anti-inflammatory or immunosuppressive agents
(systemic and topical glucocorticoids, ciclosporin, azathioprine) sometimes,
but not always, at low doses and for a short period.^94,95 However, some of the authors have seen many dogs with
CanL and pemphigus foliaceus that necessitated intense and extended
immunosuppressive treatment, which greatly interfered with the efficacy of
anti-Leishmania treatment and the prognosis. Again, concurrent
administration of azathioprine and allopurinol is contraindicated.

Conclusion: Pustular
skin disease that does not respond to the standard treatment of CanL should be
treated with glucocorticoids and perhaps other immunosuppressive drugs (SORT:
weak).

8.8. Measures against sand fly bites

            Despite
the lack of relevant studies, it is reasonable to protect dogs under treatment
from further sand fly bites that may inject new parasites and that will also
inject insect saliva, that may have local immunosuppressive effects.²⁷⁹ This is also important to reduce the risk of
transmission to other dogs and humans. Therefore, it is prudent to use, for
life, effective insect repellents in all treated dogs living in endemic areas.
Unfortunately, the efficacy of insect repellents is not absolute and not all
effectively treated dogs are necessarily unable to transfer parasites to
vectors; furthermore, if this happens, the transmitted parasites would have
been exposed to drugs and may be more likely to be drug-resistant. Under
laboratory conditions, some sand fly species (Ph. perniciosus, L.
longipalpis) fed on healthy dogs receiving isoxazolines (afoxolaner,
fluralaner) at registered dosage regimens show increased lethality within a
time frame shorter than the time required for Leishmania spp. to evolve
to the metacyclic promastigote stage and be able to infect new hosts.^280-282 Therefore, administration of isoxazolines for life may
reduce the spread of drug-resistant strains of L. infantum.

Conclusion: Effective
topical insect repellents and oral isoxazolines (afoxolaner, fluralaner) at the
registered dosage regimens are recommended for all dogs treated for CanL that
live in endemic areas (SORT: weak).

8.9. Treatment monitoring

            The
aim of close patient monitoring during the whole treatment period is to ensure
the efficacy of the selected therapeutic intervention(s), detect possible
relapses during long-term allopurinol administration, and avoid or treat
medication adverse effects. The moderate-to-strong SORT in favor of allopurinol
in combination with meglumine antimoniate or with miltefosine as first-line
treatment, and the strong SORT in favor of allopurinol-aminosidine as a
second-line treatment, were based on RCTs, reflecting the efficacy and safety
of these interventions for the average dog with CanL, the severity of the
latter depending on the inclusion criteria of each study. Therefore, efficacy
cannot be guaranteed for every treated dog, especially if we consider that drug-resistant
strains of L. infantum do exist, may become more common in the future,
and their prevalence may differ among geographical areas.^{169,198,199,218} Also, due to the low number of dogs enrolled in these RCTs and in
non-controlled trials that were used to obtain safety information, published
studies may not have been enough to capture uncommon adverse effects.

            In
addition to a detailed history, thorough physical examination and measurement
of blood pressure, the minimum laboratory examinations should evaluate those
parameters found to be associated with the response to treatment, lack of
response or relapses during treatment, in the RCTs and in open clinical trials.
These include complete blood count, serum biochemistry (including at minimum
total proteins, albumins, globulins, albumin/globulin ratio, BUN, creatinine,
iP, and ALT), serum protein electrophoresis, complete urinalysis including
measurement of UPC, quantitative serology, and evaluation of parasitic burden
(e.g., lymph node and/or bone marrow semiquantitative microscopy²⁸³ and/or qPCR).^{102,107,128,169,172,223,240,244,246,250,283-288} Blood qPCR is not considered reliable, probably due to daily variations in
circulating number of parasites, but it has the advantage of easier sampling.^102,128,169 Also, monitoring of
acute phase proteins (e.g., CRP, ceruloplasmin, ferritin), especially if their
concentration was abnormal at baseline, can provide valuable information on treatment
efficacy, and may predict future relapses.^{223,285,289,290} Furthermore, kidney and urinary bladder ultrasonography (U/S) should be
considered in allopurinol treated dogs with xanthinuria and/or clinical signs
of urolithiasis,²²⁵ and close monitoring for acute pancreatitis (e.g.,
canine specific pancreatic lipase, abdominal U/S) is advised during meglumine
antimoniate administration.²³⁹