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International Journal of Antimicrobial Agents 44 (2014) 463–465

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents

j o ur nal ho me pag e: ht tp : / /www.e lsev ier .com/ locate / i jant imicag

hort Communication

linical treatment of cutaneous leishmaniasis in dogs withurazolidone and domperidone

tela Rechinelli Passosa, Tadeu de Azevedo Rodriguesa, Ana Paula Madureirab,odolfo Cordeiro Giunchetti c, Marcos Santos Zaninia,∗

Laboratório de Microbiologia Veterinária e Zoonoses, Departamento de Medicina Veterinária, Universidade Federal do Espírito Santo, Alto Universitário,/n, Guararema, Alegre, ES CEP 29500-000, BrazilDepartamento de Engenharia de Biossistemas, da Universidade Federal de São João Del Rei, São João Del Rei, MG CEP 36301-160, BrazilLaboratório de Biologia das Interac ões Celulares, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG CEP1270-901, Brazil

r t i c l e i n f o

rticle history:eceived 10 March 2014ccepted 14 July 2014

eywords:eishmania braziliensisreatmentomperidone

a b s t r a c t

Cutaneous leishmaniasis (CL) is a zoonosis and a public health problem in countries of subtropical Amer-ica. The aim of this study was to investigate the efficacy of furazolidone and domperidone treatment ofdogs naturally infected with Leishmania (Viannia) braziliensis. Infection was confirmed by PCR and parasiteculture of tissue collected from skin scrapings of the lesion borders of dogs. Naturally infected animalswere divided into control (n = 4) and treatment (n = 8) groups. The treatment group was administeredfurazolidone for 21 days interspersed with domperidone for 10 days by oral gavage. Dogs that showed nolesion healing during this period were administered the same treatment cycle for up to 93 days. Among

urazolidoneogkin lesions

the eight treated animals, seven were clinically cured without recurrence of skin lesions during the 12-month study period. However, during lesion healing, skin scrapings were positive for L. (V.) braziliensis byPCR; no growth of the protozoan in NNN–LIT medium occurred until the end of follow-up. These resultssuggest that treatment with furazolidone and domperidone is effective for epithelialisation and lesionhealing of dogs with clinical CL caused by L. (V.) braziliensis.

© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

. Introduction

Cutaneous leishmaniasis (CL) is a public health issue in countriesf subtropical America. In Brazil, the disease occurs throughout theountry and the causative agent responsible for the largest num-er of cases is Leishmania (Viannia) braziliensis. Euthanasia of wildnd domestic animals is not accepted as a control measure forL [1]. In addition, treatment of infected animals with the drugssed in humans infected with CL is not allowed in order to avoidhe development of parasite resistance [1]; thus, clinically affectednimals live with skin lesions likely due to secondary contamina-ion that are not treated [2]. The constant search for new drugso treat leishmaniasis has led to the discovery of furazolidone [N-

5-nitro-2-furfurylidene)-3-amino-2-oxazolidinone], a nitrofuranerivative with antiprotozoal and antibacterial effects. Its leish-anicidal action and morphological activity are due to its ability to

∗ Corresponding author. Tel.: +55 2835528916;ax: +55 2899005621; +55 2835528653.

E-mail address: marcos.zanini@ufes.br (M.S. Zanini).

ttp://dx.doi.org/10.1016/j.ijantimicag.2014.07.011924-8579/© 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights

change and cause the loss of intracellular organelles targeted to thenuclear membrane and mitochondria [3]. Immunomodulation isalso needed for the treatment of leishmaniasis. This can be achievedby the use of domperidone, a gastric prokinetic with antiemeticactivity. It is a dopamine D2 receptor antagonist and hyperpro-lactinaemic drug that causes the release of serotonin, which in turnstimulates prolactin production. Prolactin appears to have a centralrole in the specific cellular immune response to leishmaniasis [4].

Thus, the aim of this study was to evaluate the efficacy of fura-zolidone interspersed with domperidone in the clinical treatmentof dogs naturally infected by L. (V.) braziliensis.

2. Materials and methods

2.1. Animals

The location of 12 dogs with characteristic CL lesions and

natural infections with L. (V.) braziliensis was indicated by theSanitary Surveillance, Municipality of Iúna (Espírito Santo State,Brazil) for use in the present study. The animals belonged to bothsexes, were 5–12 years of age and were of undefined breeds. All

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f the procedures described here were approved by the Ethicsommittee on Animal Experiments of the Universidade Federal dospírito Santo (CEUA–UFES) (Alegre, ES, Brazil).

.2. Standardisation and characterisation of the study group

Dogs were divided in two groups: a control group (n = 4) witho treatment that remained in their homes; and a treatmentroup (n = 8). Dogs in the treatment group were administered fura-olidone alternated with domperidone by oral gavage and wereaintained in a private kennel.Sixty days before starting treatment, all animals underwent a

tandardised sanitary treatment. They were dewormed with prazi-uantel, pyrantel pamoate and febantel. The animals were fed withtandard dog food (crude protein 21%) until the end of the study.

.3. Monitoring animals during treatment

The following factors were measured in dogs of the treatmentroup: complete blood count and classical clinical chemistry mark-rs of hepatotoxicity, such as aspartate aminotransferase (AST), ala-ine aminotransferase (ALT), gamma-glutamyltransferase (GGT)nd liver bilirubin. Dogs also underwent a renal biochemistry pro-le (urea, creatinine and albumin). The sizes of the skin lesions of allnimals were monitored using a calliper (Mitutoyo, Taipei, Taiwan),nd lesion size was expressed in mm2. Measurements were per-ormed for the control group on days 0, 120, 240 and 360, whilstor the treatment group the measurements were performed weeklyuring the days of medication administration in addition to weeklyeighing of the dogs for dose adjustment.

.4. Identification of L. (V.) braziliensis

For identification of L. braziliensis infection in the dogs, skincrapings of the lesion borders of dogs were performed whilehe dogs were anaesthetised with a combination of ketamine15 mg/kg intramuscular) and xylazine (2 mg/kg intramuscular).his was performed before treatment and on Days 120, 240 and60 after the beginning of the study. A portion of the scrapingsollected was used for culture and another portion was used toerform PCR. For culture, the skin scrapings were treated with

penicillin–streptomycin solution and were seeded on NNN–LITedium [5]. Cultures were kept in a refrigerated incubator at

4 ± 1 ◦C for 30 days. After this period, promastigotes of the parasiteere evaluated using light microscopy. Also, for species identifica-

ion as L. (V.) braziliensis, the skin scrapings and the cultured pro-astigotes were submitted to PCR using primers B1–B2, because

he region of residence of the dogs was only endemic to L. (V.)raziliensis [6,7]. Epithelial tissue of the healing lesions after treat-ent was also subjected to scraping (culture and PCR) to assess the

resence of protozoa by PCR and culture on Days 120, 240 and 360.

.5. Treatment

Preliminary tests were conducted on a few healthy dogs byreating them with furazolidone at 50 mg/kg/day [8] and a lowerose of 40 mg/kg/day. The dogs showed anorexia and motor inco-rdination within 5 days (unpublished data). Thus, a treatment of5 mg/kg/day furazolidone (Giarlam® tablets, 200 mg; UCI-Farmand. Farm. Ltda., São Bernardo do Campo, SP, Brazil) administeredrally, divided into two doses once every 12 h for 21 consecutiveays was chosen. This was alternated with domperidone admin-

stered orally at 1 mg/kg twice daily (Motilium® tablets, 10 mg;anssen-Cilag Farm. Ltda., São José dos Campos, SP, Brazil) for 10onsecutive days [4]. The complete treatment was carried out in1-day cycles. For dogs that did not experience any lesion healing

timicrobial Agents 44 (2014) 463–465

during this period, new cycles of treatment were administered untilthe expected effect was observed.

2.6. Statistical analysis

Fisher’s exact test was used to verify the association of epithe-lialisation and lesion healing with treatment of the animals.Mann–Whitney U-test was used to evaluate the difference betweenthe initial and final area of the lesion. Statistical analysis wasperformed using GraphPad Prism 5.0 statistical software (Graph-Pad Software Inc., San Diego, CA). All values were expressed asthe mean ± standard error of the mean or median (interquartilerange) as appropriate. The Shapiro–Wilk normality test was con-ducted to test the null hypothesis that the frequency distribution ofsampling errors follows a Gaussian distribution [9]. When the datadid not deviate from a Gaussian distribution (P > 0.05), they wereanalysed using the paired t-test, otherwise the Wilcoxon matched-pairs signed-rank test (W), Mann–Whitney non-parametric U-testor Fisher’s exact test was used as appropriate. A P-value of <0.05was regarded as statistically significant.

3. Results and discussion

3.1. Characterisation and monitoring of the study group

The adjustment period of 60 days was essential to improve thehealth and nutritional status of the dogs and resulted in a meanweight gain of 12% of the dogs’ original weights. There were asso-ciations between nutritional impairment status and age, mucosalleishmaniasis, events affecting food intake and lesion healing [10].During treatment, the dogs experienced weight loss of up to 21% oftheir body weight. As both drugs utilised cause hepatic biotransfor-mation and adrenal excretion, it was necessary to evaluate the dogs’complete blood counts and to perform liver and renal biochemistryprofiles.

Prior to treatment, the main haematological findings in thetreatment and control groups were thrombocytopenia (50% of ani-mals), eosinophilia (25%) and anaemia (8.33%), whilst a previousstudy observed 82.4%, 41.2% and 70.6%, respectively, for the samecharacteristics in dogs with CL [11]. At 120 days from the beginningof the study, one dog that had been treated and cured with fura-zolidone alternated with domperidone continued to have anaemia.Four other cured dogs presented with thrombocytopenia but noeosinophilia. At the end of 360 days, only two cured dogs stillhad thrombocytopenia. The control group maintained their initialhaematological disorders. Biochemical tests for the two groups ofdogs before treatment revealed an ALT above the normal range forfour dogs (33.3%), as well as elevated GGT for eight dogs (66.7%).Another author observed an increase in serum ALT and AST in63.63% of animals without changes in GGT values [12].

Regarding urea, two (16.7%) of the animals showed values belowthe reference, whereas previous studies reported this in 22.72%of animals. Until 120 days after treatment, the treatment groupcontinued to present with serum GGT levels above reference val-ues as well as decreased ALT and AST values only in 4/8 treateddogs. Further studies are needed to determine the biochemicaland haematological parameters of animals with symptoms of CLin order to elucidate the true effects of drugs on treated animals,considering the effects of the infection in these animals.

3.2. Identification of L. (V.) braziliensis

Scrapings of the lesion borders were seeded in NNN–LIT mediumand promastigotes from all dogs were cultured between 7 and19 days before treatment. Scrapings from the dogs of the treatmentgroup that experienced lesion healing after treatment did not show

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rotozoan growth until the end of follow-up. For the control group,rowth of promastigotes in NNN–LIT medium from four collec-ions of lesion scrapings taken during the 12-month evaluation wasbserved. PCR of the tissue scrapings of all animals (treatment andontrol groups) were positive before and after treatment. PositiveCR results for tissue scrapings were also found by other authors13].

.3. Treatment

Considering the initial poor nutritional status of the dogs, andaptation period was necessary for self-healing of the infectednimals. Among the eight treated animals, only one did not experi-nce effective epithelialisation of the mucosal lesion possibly dueo the dog’s advanced age of >12 years. Since the course of clin-cal leishmaniasis manifestations depends on the immunologicaltatus of the animal, the age of the animal may be an impor-ant factor for treatment failure; old animals present with immuneesponse impairment [10]. Still, there was mucosal lesion regres-ion in the >12-year-old dog, and three animals required more thanne cycle (31 days) of treatment for lesion healing. We suggesthat epithelialisation and lesion healing in most animals followingreatment was due to the leishmanicidal activity of furazolidonehat has previously been reported [8] to reduce clinical signs dur-ng the experimental treatment of golden hamsters infected witheishmania (Leishmania) chagasi. However, human treatment withurazolidone at a dose of 8 mg/kg/day for 10 days was not suc-essful [14]. Most likely, the action of furazolidone is enhanced byhe immunostimulatory activity of domperidone [8], since curingeishmaniasis depends on the immune status of the animal [10].owever, no lesions decreased in size during the administration ofomperidone.

In humans, persistence of this parasite was observed afterchieving a clinical cure of leishmaniasis [13]. However, lesion heal-ng and epithelialisation themselves have relevance; sandflies haveeen observed to only be infected when they blood-fed directlyrom the lesions of naturally infected dogs. When sandflies werellowed to feed on the intact skin of the same dogs, the sandflieshowed no parasite forms by xenodiagnoses [15].

.4. Statistical analysis

The mean number of days of healing of the lesions ranged from9.88 ± 10.37 days in the treatment group to 91.0 ± 0.0 days in theontrol group (unpaired t-test = 2.57, P = 0027); control animalsxperienced no regression of the lesion. This shows that, on aver-ge, two sessions (62 days) of furazolidone treatment alternatedith domperidone led to complete remission of the lesions in these

nimals.During the 21-day period of administration of furazolidone,

statistically significant lesion area reduction was observed−0.12 cm2 (–0.46 to –0.03 cm2)] between the start and end ofach cycle (Mann–Whitney U = 53.50, P = 0.004), whilst during the0 days of administration of domperidone the lesions retained theame area [–0.0 cm2 (–0.07 to –0.07 cm2)].

It was also observed that the initial and final size of the lesionfter cycles of alternating furazolidone and domperidone wasignificantly different for the treatment group [start = 0.770.25–0.98); end = 0.0 (0.0–0.0); Wilcoxon matched-pairs

[

timicrobial Agents 44 (2014) 463–465 465

signed-rank test W = 36, P = 0.008] but not for the control group[start = 1.11 (0.61–1.63); end = 0.78 (0.33–1.57); W = 2, P = 0.875].Epithelialisation of the lesion was observed in the treatment group.Also, an association between lesion healing and treatment usedwas confirmed by the results of a Fisher’s exact test (P = 0.01).

In conclusion, these data suggest that treatment with fura-zolidone, which has leishmanicidal activity, alternated withdomperidone, which has a known immunostimulatory action, iseffective for developing epithelialisation and lesion healing in dogswith clinical CL caused by L. (V.) braziliensis.

Funding: This work was supported by Fundac ão de Ampara àPesquisa do Espírito Santo (FAPES) (Grant no. 54694442/11) andCoordenac ão de Aperfeic oamento de Pessoal de Nível Superior(CAPES).

Competing interests: None declared.Ethical approval: This study was approved by the Ethics Commit-

tee on Animal Experiments of the Universidade Federal do EspíritoSanto (CEUA–UFES) (Alegre, ES, Brazil) [no. 013/2012].

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[2] Banuls AL, Bastien P, Pomares C, Arevalo J, Fisa R, Hide M. Clinical pleiomor-phism in human leishmaniases, with special mention of asymptomaticinfection. Clin Microbiol Infect 2011;17:1451–61.

[3] Reimão JQ, Taniwaki NN, Tempone AG. Furazolidone is a selective in vitro can-didate against Leishmania (L.) chagasi: an ultrastructural study. Parasitol Res2010;106:1465–9.

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