Efficacy of Combined Therapy with Liposome-Encapsulated
Meglumine Antimoniate and Allopurinol in Treatment of Canine
Sydnei M. da Silva,aIzabela F. G. Amorim,bRaul R. Ribeiro,c* Erly G. Azevedo,cCynthia Demicheli,dMaria N. Melo,aWagner L. Tafuri,b
Nelder F. Gontijo,aMarilene S. M. Michalick,aand Frédéric Frézardc
Departamento de Parasitologia,aDepartamento de Patologia,bDepartamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas,cand Departamento de Química,
Instituto de Ciências Exatas,dUniversidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were
per os) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty
worldwide. Even with scientific advances related to diagnosis,
treatment, and prevention over the past 10 years, VL still is a
neglected disease leading to ?60,000 human deaths/year. The
clinical manifestations of VL are attributed to obligatory intracel-
lular protozoa of the Leishmania donovani complex and, depend-
ing on the etiological agent, the disease presents two distinct
forms: anthroponotic VL that is endemic in India and Central
Africa, caused by L. donovani, and zoonotic VL that occurs in
caused by Leishmania infantum. Domestic dogs are the most im-
portant urban reservoirs of L. infantum, which is transmitted to
genera Lutzomyia and Phlebotomus (Diptera: Psychodidae; Phle-
botominae) in the New World and Old World, respectively (7,
33). The disease in dogs is characterized by a marked pleomor-
phism, and the clinical signs vary according to the immune re-
sponse of the animals toward the infection. In general, the main
clinical signs of canine visceral leishmaniasis (CVL) are various
degrees of dermopathy, lymphadenopathy, onychogryposis,
weight loss, abnormalities of the musculoskeletal system, eye le-
ing from immune complex deposition in tissues (e.g., vasculitis
and arthritis) (30).
The treatment of dogs affected with VL has been practiced in
Europe since the middle of the 20th century (1). Since then,
the pentavalent antimonials, including meglumine antimoniate
(MA) and sodium stibogluconate, have been the main class of
leads to high rates of morbidity and mortality in humans
treatment with antimonial drugs does not promote parasitologi-
cal cure in infected dogs, leading to frequent relapses (23) and
necessitating continuous administration of the drugs, which are
both poorly tolerated and expensive.
The purine analog allopurinol is an alternative orally active
drug (6) which presents low toxicity, is effective in reverting the
However, allopurinol also is unable to promote parasitological
cure in dogs with VL (23).
the first-line pharmaceutical protocol for CVL. The basis for the
use of this combination is the synergism between these drugs
against Leishmania parasites, as previously demonstrated in vitro
(22) and in vivo (1, 15, 24, 27). However, although most dogs
recover clinically after therapy, complete elimination of the para-
site is usually not achieved, and infected dogs may eventually re-
risk of long-term treatment for selection of Leishmania strains
Received 26 January 2012 Returned for modification 21 February 2012
Accepted 1 March 2012
Published ahead of print 12 March 2012
Address correspondence to Frédéric Frézard, firstname.lastname@example.org.
*Present address: Centro de Ciências Agrárias, Ambientais e Biológicas da
Universidade Federal do Recôncavo da Bahia, Cruz das Almas, Buenos Aires, Brazil.
S.M.d.S. and I.F.G.A. contributed equally to this work.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
aac.asm.orgAntimicrobial Agents and Chemotherapyp. 2858–2867June 2012 Volume 56 Number 6
resistant to antimonial drug, WHO strongly supports the search
for more effective therapeutic protocols (21, 23, 33).
liposome-encapsulated antimonial drugs were hundreds of times
more effective than the free drugs against experimental VL based
on parasite suppression in the liver (4). Since then, much effort
has been devoted to the search for efficacious liposomal formula-
lation of MA (LMA) was recently developed that has significant
pharmaceutical and pharmacological advantages over conven-
Sb/kg of body weight at 4-day intervals (28) promoted both long-
term parasite suppression and reduction of infectivity to sand
With the aim of further improving the treatment efficacy of
LMA in dogs, a new protocol was designed which combines LMA
with allopurinol and takes advantage of the synergistic antileish-
work, the efficacy of the combination of LMA with allopurinol as
with L. infantum has been established for the first time, through
infectivity of dogs to sand flies.
MATERIALS AND METHODS
Materials and drugs. Cholesterol and dicetylphosphate were purchased
phosphatidylcholine was obtained from Lipoid (Ludwigshafen, Ger-
many). N-Methyl-D-glucamine and antimony pentachloride (SbCl5,
99%) were obtained from Aldrich Chemical Co. (Milwaukee, WI). Allo-
purinol was purchased from a commercial laboratory for pharmaceutical
dosages of 20 mg/kg of body weight. Meglumine antimoniate (MA) was
synthesized, as previously described (14), from equimolar amounts of
N-methyl-D-glucamine and pentavalent antimony oxyhydrate freshly
prepared from the hydrolysis of antimony pentachloride in water. The
resulting product contained 28% Sb by weight. As previously established
(18), synthetic MA may be replaced by commercial MA (Glucantime;
Sanofi-Aventis Farmacêutica Ltda., São Paulo, Brazil) in the preparation
of the liposomal formulation.
Animals. The present study was approved by the Ethical Committee
ing to the international guidelines (Principles of Laboratory Animal Care
kg (mean ? standard deviation), of unknown age, and naturally infected
an area where canine visceral leishmaniasis is endemic. These dogs were
ities of the municipality’s Visceral Leishmaniasis Control Program.
the Institute of Biological Sciences (ICB), Federal University of Minas
Gerais (UFMG) using indirect immunofluorescence assay (IFAT) and
enzyme-linked immunosorbent assay (ELISA) (5). All animals were
?0.100; 1:400 dilution). In addition, specific PCR of bone marrow aspi-
rate was used to confirm the infection of the animals by L. infantum (13).
drinking water and balanced commercial food (Nero Refeição; Total Ali-
treatment, dogs were treated against intestinal helminths (Helfine cães;
Agener União, Brazil) and ectoparasite infestations (Frontiline Top Spot;
Merial, Brazil) and immunized against rabies (Defensor; Pfizer Saúde
Animal, Brazil) and other infectious diseases (Vanguard HTLP 5/CV-L;
Pfizer Saúde Animal, Brazil).
lated meglumine antimoniate (LMA) was prepared as previously de-
scribed (29). Briefly, small unilamellar vesicles (SUVs) were obtained by
mixing distearoylphosphatidylcholine, cholesterol, and dicetylphosphate
(molar ratio of 5:4:1) following ultrasonication in deionized water at a
final lipid concentration of 55 g/liter. Then, the SUV suspension was fil-
tered with a sterile 0.22-?m membrane and mixed with sucrose (sugar/
ing mixture was immediately frozen in liquid nitrogen and subsequently
dried (freeze dryer, 4.5 liters; Labconco, United Kingdom). At this point,
6 months without any change of final vesicle size and drug encapsulation
efficiency characteristics. The day before administration, the lyophilized
powder was rehydrated with a solution of MA in water (antimony con-
centration of 0.65 M, corresponding to 40% of the original SUV volume)
and the resulting suspension was vortexed and incubated for 30 min at
55°C. Then, the same volume of phosphate-buffered saline (PBS; 0.15 M
NaCl, 0.01 M phosphate, pH 7.2) was added to the mixture, followed by
vortexing and incubation for 30 min at 55°C. The resulting liposome
suspension was diluted in PBS and centrifuged (25,000 ? g for 30 min at
ugation, the liposome pellet was washed twice with isotonic saline and
resuspended in sterile saline solution, and the final antimony concentra-
determined by atomic absorption spectroscopy (AA600; PerkinElmer,
Instruments, United Kingdom). In parallel, lyophilized SUVs were rehy-
of MA, using the same method as described above. The final suspension,
dynamic vesicle diameter and polydispersity index.
as follows. In the LMA?Allop group, eight animals were treated with six
with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day
intervals. In the Allop group, eight animals were treated with allopurinol
(20 mg/kg of body weight/24 h per os) for 140 days and six doses of
isotonic saline given at the same volume and time intervals as LMA in the
LMA?Allop group. In the LEMP?Allop group, eight animals were
treated with six doses of empty liposomes given at the same volume and
time intervals as LMA in the LMA?Allop group and allopurinol (20
mg/kg of body weight/24 h per os) for 140 days. In the LEMP group, eight
animals were treated with six doses of empty liposomes given at the same
12 animals received six doses of isotonic saline given at the same volume
and time intervals as LMA in the LMA?Allop group.
Following day 140 after the beginning of treatment, all animals of the
six experimental groups were kept in the kennel for 60 days without any
intervention, representing a total experimental period of 200 days. Ani-
mals were clinically monitored during the experimental period and were
submitted to clinical and parasitological evaluations just before and on
Liposomal Meglumine Antimoniate plus Allopurinol
June 2012 Volume 56 Number 6aac.asm.org 2859
During the experimental period, seven animals died: two dogs in the
LMA?Allop group, two in the LMA group, and one in the LEMP group,
one in the LEMP group), most probably due to the natural evolution of
Clinical evaluation. Just before treatment and on days 140 and 200
after the start of treatment, the dogs were inspected for the presence of
clinical signs of CVL, and serum and plasma (using EDTA as an antico-
tibodies (IFAT), hemogram, and levels of serum urea, creatinine, alanine
proteins, globulins, albumins, and albumin/globulin (A/G) ratio.
Based on the results of the physical examination, levels of anti-Leish-
mania antibodies determined by IFAT (5), and laboratory findings in
score 0 corresponds to absence of clinical signs of CVL, negative serology
(IFAT ? 1:40), and no abnormalities in hemogram and serum biochem-
istry, and score values from 1 to 4 correspond to the mild, moderate,
severe, and extremely severe disease clinical stage, respectively.
Parasitological evaluation. Quantitative real-time PCR (qPCR) was
used to determine the parasite loads in bone marrow and spleen just
before treatment and on days 140 and 200 after the start of treatment.
Dogs were submitted to general anesthesia using the combination of 2
mg/kg of body weight of xylazine chloridrate (Calmium; União Química
ridrate (Ketamina Agener; União Química Farmacêutica S/A, Brazil) by
collected, followed by 1.0 ml of spleen aspirate after previous localization
of the organ with portable ultrasound equipment (SonoSite SonoHeart
Elite Superior 180 Plus; SonoSite Inc., United States). At euthanasia (day
of the right ear were collected to perform qPCR, in addition to the bone
marrow and spleen aspirates. Skin samples were also obtained just before
treatment to be used as controls. All samples were stored at ?80°C until
required for further processing.
blood and tissue kit (Qiagen, Inc., United States), used according to the
ers (forward, 5= TGT CGC TTG CAG ACC AGA TG 3=, and reverse, 5=
accession number AF009147) were used (9). PCR was carried out with a
final reaction mixture volume of 25 ?l containing 200 nM forward and
reverse primers, 1? SYBR Green PCR master mix (Applied Biosystems,
United States), and 5 ?l of template DNA. The PCR conditions were as
follows: an initial denaturation step at 95°C for 10 min, followed by 40
1 min. Standard curves were prepared for each run using 10-fold serial
of pGEM-T Easy vector system plasmids (Promega, United States) con-
using primers that amplified a 307-bp fragment (forward, 5= CTT CTA
CAA CGA GCT GCG CG 3=, and reverse, 5= TCA TGA GGT AGT CGG
TCA GG 3=) in order to verify the integrity of the samples and to normal-
ize the initial concentrations of DNA. The number of copies of L. infan-
tum in the samples was adjusted using the ?-actin correction factor ob-
close to 100% and the standard curves presented correlation coefficients
between plasmid concentrations and threshold cycle (CT) values ranging
from 0.97 to 0.99. In our assays, standard curves allowed the detection of
a limit of 0.8 parasites/ml of bone marrow and 0.25, 0.84, and 1.1 para-
sites/mg of liver, spleen, and skin, respectively. The reaction mixtures
were processed and analyzed in the ABI Prism 7500 sequence detection
system (Applied Biosystems, United States).
Bone marrow aspirates (1.0 ml) were collected on day 200 and imme-
Nicole) medium, enriched with minimum essential medium (MEM ?;
Gibco, United States). Cultures were maintained at 23 ? 1°C and exam-
ined each 10 days for 30 days in order to identify the presence of promas-
Xenodiagnosis. Xenodiagnosis was performed as previously de-
scribed (12), with some modifications, in order to verify the ability of
treated dogs to infect the sand flies. Briefly, just before treatment and on
TABLE 1 Staging system used for classification of dogs with visceral leishmaniasis
Clinical stage IFAT titer Clinical signsClinicopathological abnormalities Score
Stage I: mild disease Negative or
Peripheral lymphadenopathy and/or one
slight dermatological alteration, such
as papular dermatitis, exfoliative
dermatitis, seborrheic dermatitis, or
Peripheral lymphadenopathy associated
with two or more signs, as follows:
cutaneous alterations (papular
dermatitis, exfoliative dermatitis,
onychogryposis, or ulcerative
dermatitis), anorexia, mucopurulent
fever, and epistaxis
Clinical signs of stage II and lesions
associated with immune complex,
such as vasculitis, arthritis, and uveitis
No clinicopathological abnormalities, creatinine
?1.4 mg/dl, urea ?25 mg/dl, and A/G ratio
Stage II: moderate disease
?1:80Mild nonregenerative anemia and/or
hypoalbuminemia, creatinine ?1.4 mg/dl,
urea ?25 mg/dl, and A/G ratio ?0.6
Stage III: severe disease
?1:160Mild nonregenerative anemia and
hypoalbuminemia, creatinine 1.4-2 mg/dl,
urea ?25 mg/dl, and A/G ratio ?0.6
Nonregenerative anemia and
hypoalbuminemia, creatinine ?2 mg/dl, urea
?25 mg/dl, and A/G ratio ?0.6
Stage IV: very severe
?1:640Clinical signs of stage III and signs of
chronic kidney disease, such as
nephrotic syndrome and uremic crisis
da Silva et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
days 140 and 200 after the start of treatment, animals were submitted to
general anesthesia as described above and the internal surface of the right
from the colony of the Laboratory of Physiology of Haematophagous
feed directly on the right ear for 30 min in a dark room. After the blood
water and kept at 28°C in the insectary for 5 days. On the fifth day, the
females of L. longipalpis were dissected in a drop of PBS solution and
midguts were examined under an optical microscope at 400? magnifica-
tion to verify the presence of promastigote forms and to determine the
infection ratio. The estimated number of promastigotes in each midgut
was determined as follows: ?, absence of promastigotes; ?, presence of 1
to 50 promastigotes; ??, 51 to 200 promastigotes; and ???, ?201
Cure criteria. The criteria established to consider an animal as cured
of CVL on day 200 after the start of treatment, independent of the treat-
ment protocol received, were (i) absence of parasites in bone marrow
aspirate culture, (ii) negative results for parasitological evaluations by
GraphPad Prism version 5.00 for Windows (GraphPad Software, United
States). According to the Kolmogorov-Smirnov test, experimental data
were not normally distributed. All data were not normally distributed.
Then, the Kruskal-Wallis test followed by Dunn’s multiple comparison
test or the Mann-Whitney test was used to compare clinical stage and
tissue parasitic loads. The Friedman test was used to compare the clinical
stages and the tissue parasite loads in each group just before and on days
140 and 200 after the start of treatment. Comparison of qPCR results for
skin samples before and after treatment was performed using Wilcoxon
matched pairs. Fisher’s exact test was used to compare the sand fly infec-
tion efficiencies and the proportions of cured dogs between different
groups. A significance level of 95% was applied in all statistical tests.
Treatment of mongrel dogs naturally infected with Leishmania
infantum was performed with six doses of LMA (6.5 mg Sb/kg/
dose) given at 4-day intervals plus allopurinol (20 mg/kg/24 h per
of clinical and parasitological parameters determined just before
treatment and on days 140 and 200 after the start of treatment.
Comparison was performed with experimental groups treated
nol (LEMP?Allop). Control groups received either saline or
empty liposomes (LEMP).
Clinical parameters. Before treatment, all animals presented
clinical signs of CVL, as established by the inclusion criteria. Der-
matological alterations, such as exfoliative dermatitis, seborrheic
dermatitis, localized alopecia, ulcerative dermatitis over joint
prominences, and onychogryposis, were the main clinical signs
signs were lymphadenopathy and mucopurulent conjunctivitis/
bocytopenia (23.0%), leukocytosis (24.5%), serum urea (84.6%),
and globulins (32.6%) above and A/G ratio (69.2%) below the
reference values were the main abnormalities found in hemo-
grams and serum biochemistry of the animals. All animals were
also positive according to IFAT, and 95% of these dogs presented
high levels of anti-Leishmania antibody titers (?1:640).
Taking into account clinical, laboratory, and serological data
and a recently proposed clinical classification of CVL (30), each
animal received a specific score, the highest score corresponding
to the most severe pattern of the disease. The distribution of ani-
and 40.4% in stage III.
Treatment of infected dogs with LMA in combination with
allopurinol (LMA?Allop) promoted a marked reduction in anti-
determined by IFAT endpoint, showed a 20.3-fold reduction in
antibody titer, ranging from 1:8,133 before treatment to 1:400 on
reductions in the other groups were as follows: LMA, 1.9-fold
(from 1:3,867 to 1:2,000); Allop, 1.3-fold (from 1:3,940 to
1.4-fold (from 1:5,013 to 1:3,627); and saline, 2.5-fold (from
1:6,749 to 1:2,755). Interestingly, only in the LMA?Allop group
than prior to treatment (P ? 0.05; Friedman).
Figure 1 compares the more general and predictive clinical
stage parameters between the different experimental groups,
taking into account the results of quantitative serology, physi-
cal examination, and clinicopathological findings. The data in-
dicate that the LMA?Allop, LMA, and Allop groups presented
significantly lower clinical scores (severity of stages) on days
140 and 200 than control groups (LEMP and saline) (P ? 0.05;
Among the different treatment protocols, the LMA-allopuri-
tion in the median clinical score of the LMA?Allop group was
significant on days 140 and 200 compared to the median score
before treatment (P ? 0.05; Friedman). Importantly, all the ani-
mals of this group (100%) presented a lower clinical score at both
times than prior to treatment. At day 200, two dogs were com-
pletely asymptomatic, with no alterations in physical examina-
tion, normal hemogram and serum biochemistry, and negative
serology (score ? 0). Another dog was classified as stage I, show-
ing no clinicopathological alterations but medium levels of anti-
of this group were classified in clinical stage II. They presented
medium levels of anti-Leishmania antibody titers (IFAT, 1:320 to
1:640) and, associated with this alteration, one dog had a low
platelet level, another showed seborrheic dermatitis and lymph-
adenopathy, and the third presented a low platelet level, hy-
poalbuminemia, and a low A/G ratio (data not shown).
The LMA group presented a significant reduction in the me-
pretreatment period (P ? 0.05; Friedman). At day 200, 83.3% of
and the animals were classified as stage I (50%) or stage II (50%).
The Allop and LEMP?Allop groups presented an improve-
cal difference was found when comparing the median clinical
scores between the three time points.
the pretreatment period. In these animals, both a progressive in-
ical examination and worsening of hemogram and serum bio-
Liposomal Meglumine Antimoniate plus Allopurinol
June 2012 Volume 56 Number 6 aac.asm.org 2861
chemistry parameters were observed. Saline-treated animals
presented a significantly greater median clinical score on day 200
than before treatment (P ? 0.05; Friedman).
asitological evaluation of dogs by qPCR indicated that the LMA-
allopurinol combined treatment was the most effective protocol
for reducing the parasite burden in bone marrow and spleen. As
illustrated in Fig. 2, this combination promoted a significant re-
duction in bone marrow parasite loads on days 140 and 200 com-
presented with significantly lower parasite loads in the spleen on
days 140 and 200 than the LEMP and saline groups (P ? 0.05;
Kruskal-Wallis) (Fig. 2).
The effectiveness of the LMA-allopurinol combination for re-
ducing the parasite burden in the bone marrow and spleen was
in both tissues were significantly reduced on days 140 and 200
compared to the burdens in the pretreatment period (P ? 0.05;
Friedman). On day 140, five animals (83.3%) were negative by
qPCR in both the bone marrow and spleen. At the end of the
experimental period (day 200), three of these animals remained
negative in both the spleen and bone marrow. Another dog was
still negative only in the spleen. The median numbers of parasites
in bone marrow and spleen aspirates, as determined by qPCR on
day 140, were about 770 and 245 times lower, respectively, than
before treatment. This substantial reduction in the parasite load
was still observed at the end of the experimental period (parasite
burdens were about 660 and 104 times lower, respectively).
The LMA and Allop groups showed results similar to those for
the LMA?Allop group with respect to the reduction of parasite
day 200, treatment with LMA or allopurinol (with or without
marrow parasite burden compared to that in the pretreatment
period, confirming the inability of these drugs alone to maintain
low parasite levels in this tissue after interruption of treatment.
Parasitological evaluations in the liver. The parasite loads in
determined by qPCR. Significant parasite suppression in the liver
of dogs in comparison to the parasite suppression in control
groups was achieved in the groups that received LMA (with or
without allopurinol) but not in those receiving allopurinol with-
out LMA. All animals (100%) treated with the LMA-allopurinol
the parasite burdens in the livers of the LMA?Allop group were
significantly lower than the parasite burdens in the LEMP and
saline groups (P ? 0.05; Kruskal-Wallis). In the LMA group, five
animals (87.3%) were found to be negative and the median num-
ber of parasites in this organ was significantly lower than that in
the saline group (P ? 0.05; Kruskal-Wallis).
FIG 1 Clinical staging of dogs naturally infected with Leishmania (L.) infan-
tum before and after treatment with liposomal meglumine antimoniate
(LMA), allopurinol (Allop), empty liposomes (LEMP), or the LMA?Allop or
on days 140 and 200, respectively, after the start of treatment. LMA (6.5 mg
Sb/kg/dose), empty liposomes (same dose of lipid), and saline (same volume)
were given intravenously as six doses at 4-day intervals. Allopurinol was given
at 20 mg/kg/24 h per os for 140 days. Clinical scores are defined in detail in
Table 1. Score 0, absence of clinical signs and clinicopathological alterations
suggestive of CVL and negative serology; score 1, mild clinical stage; score 2,
moderate stage; score 3, severe stage; score 4, extremely severe disease stage.
(n ? 6 to 11). *(e,f) and *(f), P ? 0.05 according to Kruskal-Wallis test fol-
lowed by Dunn’s multiple comparison test, for comparison with LEMP (e)
and/or saline (f).
da Silva et al.
aac.asm.org Antimicrobial Agents and Chemotherapy
and on day 200 after the start of treatment. No statistical difference
corresponding to the highest proportion of negative dogs among all
FIG 2 Parasite burdens in the bone marrow (A, B, and C) and spleen (D, E, and F) of dogs naturally infected with Leishmania (L.) infantum before and after
treatment with liposomal meglumine antimoniate (LMA), allopurinol (Allop), empty liposomes (LEMP), or the LMA?Allop or LEMP?Allop combination.
of lipid), and saline (same volume) were given intravenously as six doses at 4-day intervals. Allopurinol was given at 20 mg/kg/24 h per os for 140 days, starting
for comparison with LEMP?Allop (d), LEMP (e), and/or saline (f).
Liposomal Meglumine Antimoniate plus Allopurinol
June 2012 Volume 56 Number 6aac.asm.org 2863
median number of parasites decreased about 314-fold (from 69,776
Xenodiagnosis. The effect of the treatment of dogs on their
infectivity to sand flies was investigated through xenodiagnosis in
200 after the start of treatment, using females of Lutzomyia longi-
palpis. As shown in Table 2, the LMA?Allop and Allop groups
exhibited the best results in the xenodiagnosis evaluations.
In the LMA?Allop group, three animals (50%) infected sand
the ratio of infected to fed sand flies in positive dogs), whereas
of infected sand flies were observed on both day 140 and 200
compared to the proportions in the pretreatment period (P ?
0.05; Fisher’s exact test).
Among the other groups, the Allop but not the LMA and
In the LMA, Allop, and LEMP?Allop groups, significant reduc-
were also observed compared to the proportions in the pretreat-
ment period (P ? 0.05; Fisher’s exact test) (Table 2). This is in
contrast with the LEMP and saline groups, in which the propor-
and 200 compared to the proportions in the pretreatment period.
Parasitological cure. Each animal was evaluated on day 200
using different parasitological tests, including culture from bone
marrow aspirates, qPCR of bone marrow, spleen, liver, and skin
in all the parasitological tests were considered cured of CVL. Ac-
cording to these cure criteria, the LMA-allopurinol combination
promoted parasitological cure in 50% of treated animals. An ad-
ditional immunohistochemistry test was performed, as described
previously (28), in those tissues tested by qPCR. Strikingly, nega-
tive results were obtained in all samples collected from the six
animals of this group (data not shown). Among the other five
experimental groups, parasitological cure was achieved in only
one animal that was treated with allopurinol.
As illustrated in Fig. 5, the proportion of cured dogs achieved
after treatment with the LMA-allopurinol combination was sig-
nificantly greater than that in the saline group (P ? 0.05; Fisher’s
With the aim of further improving the treatment efficacy of CVL
tic regimen was designed which combines this formulation with
allopurinol, a well-tolerated and effective leishmaniostatic drug
capable of reaching all infected sites. In comparison to our previ-
ous protocol (28), LMA was given at the same dosage (6.5 mg
Sb/kg/dose) and time intervals, but animals received six doses
instead of four. Thus, a more effective protocol was also expected
(L.) infantum after treatment with liposomal meglumine antimoniate (LMA),
allopurinol (Allop), empty liposomes (LEMP), or the LMA?Allop or
LEMP?Allop combination. LMA (6.5 mg Sb/kg/dose), empty liposomes
doses at 4-day intervals. Allopurinol was given at 20 mg/kg/24 h per os for 140
test, for comparison with LEMP (e) and/or saline (f).
FIG 4 Parasite burdens in the ear skin of dogs naturally infected with Leishmania (L.) infantum before and after treatment with liposomal meglumine
antimoniate (LMA), allopurinol (Allop), empty liposomes (LEMP), or the LMA?Allop or LEMP?Allop combination. Parasite burdens prior to treatment (A)
and at day 200 (B) are shown. LMA (6.5 mg Sb/kg/dose), empty liposomes (same dose of lipid), and saline (same volume) were given intravenously as six doses
at 4-day intervals. Allopurinol was given at 20 mg/kg/24 h per os for 140 days, starting from the first dose of LMA. Data are shown as dot plots, and lines
da Silva et al.
aac.asm.orgAntimicrobial Agents and Chemotherapy
for the administration of allopurinol (20 mg/kg/24 h) differed
12 h was used (15, 20). Animals also remained without any ther-
apeutic intervention from day 140 to 200, to uncover possible
relapse of CVL.
Analysis of the clinical, parasitological, and xenodiagnosis
nol, since animals treated with this protocol presented much bet-
ter clinical and parasitological profiles than those treated with
each drug alone.
Among the six experimental groups, the LMA?Allop group
was the one that exhibited the most pronounced improvement of
clinical signs and clinicopathological parameters. However, the
improvements were gradual and improvements were also ob-
served in the groups treated with LMA or allopurinol alone. Dur-
ing the experimental period, most of the animals (72% ? 19%)
sion or marked reduction in the number and degree of skin le-
sions, absence of lymphadenopathy and eye lesions, and altera-
tions in the color of the mucous membranes (data not shown).
and was also accompanied by the normalization of most hemo-
gram and serum biochemistry parameters, such as protein elec-
trophoresis, A/G ratio, number of erythrocytes, hemoglobin con-
centration, hematocrit, and platelets (data not shown).
toward improvement was observed only in the LMA?Allop
group, suggesting that, besides reversion of the physical and clin-
icopathological abnormalities, the drug combination promoted a
long-term action resulting in the prevention of disease relapse. In
contrast, the LMA, Allop, and LEMP?Allop groups presented a
slight worsening of their clinical status from day 140 to 200. This
fact can be attributed to the inability of allopurinol to prevent
relapse after interruption of its use (10, 27) and to the transitory
positive effect of LMA on the same parameters, as previously de-
scribed by our group (28).
The clinical improvement observed in the LMA?Allop group
was accompanied by a significant reduction in the parasitic load,
and specificity of the technique for the absolute quantification of
parasites (25). The LMA-allopurinol combination had the great-
est impact on parasite burden, reducing by hundreds of times the
numbers of parasites in bone marrow, spleen, and skin following
TABLE 2 Frequency of positive dogs in xenodiagnosis and proportion and intensity of infection of Lutzomyia longipalpis fed on dogs naturally
infected with Leishmania (L.) infantum before and after treatment
Frequency of positive dogs in
Proportion of infected sand flies
Intensity of infection (%)d
treatmentDay 140 Day 200
Prior to treatmentDay 140Day 200
? ?? ???? ?????? ?? ???
aLiposomal meglumine antimoniate (LMA; 6.5 mg Sb/kg/dose), empty liposomes (LEMP; same dose of lipid), and saline (same volume) were given intravenously as six doses at
4-day intervals; allopurinol (Allop) was given at 20 mg/kg/24 h per os for 140 days, starting from the first dose of LMA.
bProportions of dogs in each group whose promastigotes were identified in the midgut of Lutzomyia longipalpis females 5 days after their blood meal on the internal surface of the
right ear of these animals.
cProportions of infected Lutzomyia longipalpis females in relation to total numbers of insects dissected 5 days after their blood meal on each experimental group of dogs.
dDistribution of midgut infections according to the estimated numbers of promastigotes, categorized as follows: ?, 1 to 50 promastigotes; ??, 51 to 200 promastigotes; and
???, ?201 promastigotes.
eP ? 0.05 according to Fisher’s exact test, showing a significantly lower proportion of infected sand flies than in the pretreatment period.
fP ? 0.05 according to Fisher’s exact test, showing a significantly greater proportion of infected sand flies than in the pretreatment period.
FIG 5 Proportions of dogs cured of Leishmania (L.) infantum infection after
treatment with liposomal meglumine antimoniate (LMA), allopurinol (Al-
lop), empty liposomes (LEMP), or the LMA?Allop or LEMP?Allop combi-
nation. LMA (6.5 mg Sb/kg/dose), empty liposomes (same dose of lipid), and
saline (same volume) were given intravenously as six doses at 4-day intervals.
Allopurinol was given at 20 mg/kg/24 h per os for 140 days, starting from the
first dose of LMA. Dogs were considered cured when they showed negative
results in all the parasitological tests performed on day 200, including culture
from bone marrow aspirates, qPCR of bone marrow, spleen, liver, and skin,
and xenodiagnosis. *, P ? 0.05 according to Fisher’s exact test.
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June 2012 Volume 56 Number 6 aac.asm.org 2865
Importantly, treatment with the drug combination resulted in
spleen on days 140 and 200 compared to the parasite loads in the
pretreatment period. This is in contrast with the other groups,
which did not show significant reductions in the bone marrow
parasite loads on day 200. Furthermore, the drug combination
reduced the parasite loads in bone marrow to a significantly
taken together, indicate at least additive effects of LMA and allo-
The importance of the use of splenic aspirates in monitoring
treatment was previously demonstrated in dogs infected with Eh-
rlichia canis treated with doxycycline (19). To the best of our
knowledge, the present study is the first to evaluate the efficacy of
treatment of CVL according to the parasite burden in the spleen
the context of CVL (31), evaluation of the parasite burden in this
organ during treatment could help in monitoring relapses and
response to therapeutics.
A major benefit of LMA-allopurinol combination is its ability
to produce negative qPCR results in the liver of all treated dogs.
accumulation of antimony in the liver promoted by the liposome
group showed only one animal (18.7%) with positive qPCR of
37.5 and 62.5% positive animals.
Because of their hematophagic behavior, sand flies need direct
noninfected sand flies and from infected sand flies to noninfected
dogs, which spreads the disease to other dogs and humans (33).
Therefore, one of the most important objectives of the treat-
inating parasites in the skin or, at least, reducing the number of
parasites to such a level that transmission does not occur (24).
Thus, some authors (24) have proposed reduction in the infectiv-
ity of dogs to sand flies through treatment as a key measure in
control programs designed to eradicate active foci of CVL.
infantum after feeding in infected dogs was investigated through
for assessing the epidemiologic impact of treatment of CVL, xe-
to research institutions (11, 24). Since our data show a positive
correlation between the xenodiagnosis result and the number of
parasites in the ear skin (Spearman’s correlation, 0.6518; P ?
0.0001), in accordance with a recent study (5), skin qPCR may be
used as an alternative protocol to assess the infectivity of dogs to
the sand flies.
Both the xenodiagnosis and skin parasite load results showed
that the LMA-allopurinol combination was the most effective
protocol for inhibiting the transmission of skin parasites to L.
longipalpis. Indeed, it promoted the blockade of transmission of
highest percentage (83.3%) of dogs negative according to skin
qPCR. This is in contrast with the results obtained with the LMA
on day 200 and lower percentages of negative dogs (50 and 25%,
An important question to be answered is whether LMA-allo-
of visceral leishmaniasis, since no fully effective treatment has
the transmission of the parasite to sand flies and humans and
reduce the risk of emergence of resistance to antimony. Based on
and negative xenodiagnosis on day 200, the LMA-allopurinol
the dogs treated. In the other groups, cured animals were found
only in the Allop group, but at a much lower rate (8%). These
mote parasitological cure (28).
The criteria used here for parasitological cure are based on the
absence of parasites in critical sites of infection and on the block-
ade of parasite transmission to the sand flies at a specific time.
60-day period without treatment, to allow the occurrence of pos-
positive on day 200. In this context, our claim for achievement of
parasitological cure should be taken cautiously, since not all tis-
sues were evaluated and one cannot completely exclude the pos-
sibility of other relapses after a longer period of time without
a new, highly effective therapeutic alternative for CVL, based on
the combination of nanotechnology and conventional therapy,
ies, new protocols based on this combination should be designed
to confirm the achievement of cure in treated dogs and to further
enhance the cure rate of CVL. A higher cure rate may be expected
from an increase in the number of doses of LMA, from the use of
allopurinol for a more prolonged period of time, and from the
improvement of the liposomal formulation for more effectively
reaching less accessible sites of infection.
We acknowledge the Brazilian agencies CNPq (grants 303046/2009-0,
473534/2010-0, and 473601/2009-5), FAPEMIG (grants REDE–221/08,
REDE–40/11, APQ–01935-09, APQ–01355-09, PRONEX 2009, and
PPM–00382-11), and CAPES for financial support. R.R.R. was the recip-
ient of a postdoctoral fellowship from FAPEMIG.
helpful assistance with qPCR assays.
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