Diagnóstico da Leishmaniose Cutânea Canina

Introduction

Canine leishmaniasis is a complex disease caused by different protozoan species of the genus Leishmania, which are transmitted by phlebotomine sand flies of the genera Lutzomyia (New World) and Phlebotomus (Old World). In South America, dogs have been found infected by Leishmania amazonensis, Leishmania braziliensis, Leishmania colombiensis, Leishmania infantum (syn. Leishmania chagasi), Leishmania mexicana, Leishmania panamensis, Leishmania peruviana, and Leishmania pifanoi.¹ Nonetheless, L. infantum and L. braziliensis are the most common species infecting dogs in this region.

From a clinical point of view, L. infantum is the principal causative agent of canine visceral leishmaniasis worldwide, although other parasites have been associated with visceral disease in dogs, including L. amazonensis and L. colombiensis.¹ On the other hand, canine cutaneous leishmaniasis is mainly caused by L. braziliensis, but cases associated to other species (e.g., L. panamensis)² appear to be more common than currently recognized, in those areas where they are also known to occur in humans.

Because the clinical signs and clinico-pathological abnormalities occurring in dogs with leishmaniasis are not easily attributable to a single Leishmania species, laboratory tests should be used to confirm the diagnosis. In this context, the objective of this seminar is to describe, from a practical perspective, the diagnostic methods that veterinary practitioners may use in the diagnostic workup of canine cutaneous leishmaniasis.

Clinical and Haematological Findings

Clinically, canine cutaneous leishmaniasis is mainly characterized by nodules and ulcers, which are mainly localized on the muzzle, ears, scrotum, and feet.^2,3 However, these clinical signs may also occur in other conditions and should therefore be considered as suggestive of cutaneous leishmaniasis in dogs living in endemic areas.

There is little information on the usefulness of clinico-pathological data in the diagnostic workup of canine cutaneous leishmaniasis.^3,4 A study reported that thrombocytopaenia and anaemia were frequent findings among 17 dogs naturally infected by L. braziliensis,³ but it is unlikely that these alterations are primarily caused by this parasite. In addition, considering that these findings are unspecific and commonly seen in other diseases, their usefulness in the diagnostic workup of canine cutaneous leishmaniasis is probably negligible.

Parasitological Diagnosis

Parasitological examination is a routinely used tool for diagnosing Leishmania spp. infection in dogs. Basically, it consists of the following steps: sample collection, staining, and examination under a light microscope. Samples may be taken from bone marrow, enlarged lymph nodes, spleen, liver and even skin lesions. These samples may be obtained using a sterile needle and syringe, after proper cleanup of the area to be biopsied. Imprint/exfoliative cytology may also be performed if skin ulcers are present. Stained slides are prepared using ordinary staining methods and then examined under a light microscope (x1000 magnification). The advantages of this technique are that it does not require sophisticate equipment or expensive reagents. However, it may be time-consuming, particularly if the laboratory is analysing a large number of samples, and may lack sensitivity, especially in infected, but healthy dogs. In the same way, different Leishmania spp. cannot be distinguished based on morphology only, so the finding of amastigotes in any of the above-mentioned tissues should be reported as "positive for Leishmania spp."

Other parasitological methods include microscopic visualization of the parasites in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture. These methods are not always available in private veterinary clinics or laboratories, but have been validated by different research groups and may be useful in some situations.

Serological Diagnosis

Numerous commercial serological tests are available worldwide, including indirect immunofluorescence assays (IFA), enzyme-linked immunosorbent assays (ELISA) and rapid immunochromatographic tests. These serological tests are designed to detect anti-Leishmania spp. antibodies in dogs, presenting variable sensitivity and specificity, sometimes over 90% in both cases. These tests mostly employ L. infantum antigens (crude, purified or recombinant), but also other parasite species or strains (e.g., Leishmania major-like strain used in some tests in Brazil). Nonetheless, cross-reactivity between antibodies directed to different Leishmania spp. is the rule, not the exception. Therefore, results should be reported as "positive to anti-Leishmania spp. antibodies".

Although the detection of anti-Leishmania antibodies does not mean active infection, high antibodies titters (4-fold higher than the cut-off positive value) may be a good indicator of active infection and disease, if suggestive clinical signs are noticeable.

Molecular Diagnosis

Molecular tools based on the polymerase chain reaction (PCR) are now widely available in some countries as a routine tool for the diagnosis of Leishmania spp. infection in dogs. Different PCR protocols have been standardized to differentiate Leishmania spp. or subgenera, but some of these approaches (e.g., RFLP-PCR and real time-PCR with high resolution melt analysis)^5,6 are mostly restricted to veterinary laboratories in large urban centres, research institutes and universities.

PCR-based methods have several advantages over traditional methods. They are usually highly sensitive and highly specific. However, it is very important to underline that the general performance of a PCR may vary according to the type of sample (e.g., bone marrow and lymph nodes are preferred over blood samples), DNA extraction method (e.g., commercial kits are usually more reliable than in-house methods), and PCR protocol, including target DNA (e.g., high-copy versus single-copy genes) and reaction conditions (e.g., conventional versus real time-PCR).

Depending on the protocol used, it may "suggest" or "confirm" an infection by a given Leishmania species. In the same way, it may also provide an estimation of the infection load, if the real time-PCR is used, for example. The most important thing while requesting a PCR testing from a private veterinary laboratory is to ask exactly the question: "What does a positive result mean?"

Conclusion

Veterinary practitioners working in areas where different Leishmania spp. may infect dogs,⁷ as it is the case of several South American countries, should use proper diagnostic tools to achieve a definitive diagnosis. Once the diagnosis of canine cutaneous leishmaniasis is confirmed, the veterinary practitioner should adopt a therapeutic strategy according to current protocols and drugs available in their countries. For instance, a recent study suggested that oral administration of furazolidone for 21 days interspersed with domperidone for 10 days is effective for epithelialisation and lesion healing of dogs with cutaneous leishmaniasis by L. braziliensis.⁸ In conclusion, veterinary practitioners may decide to use parasitological, serological, and/or molecular tests in the diagnostic workup of canine cutaneous leishmaniasis,⁹ but for those based in areas where different Leishmania spp. may infect dogs, it is imperative to use molecular tools to assess the specific identity of the parasite, before taking further decisions regarding treatment and control. A misdiagnosis may have serious consequences in countries like Brazil, where dogs infected by L. infantum should be eliminated, according to current recommendations from the Brazilian Ministry of Health. Indeed, a study conducted 10 years ago already demonstrated that many seropositive dogs that are eliminated due to a "suspected L. infantum infection" are, actually, infected by L. braziliensis.¹⁰

References

1.  Dantas-Torres F. Canine leishmaniosis in South America. Parasit Vectors. 2009;2(Suppl 1):S1.

2.  Vélez ID, Carrillo LM, López L, Rodríguez E, Robledo SM. An epidemic outbreak of canine cutaneous leishmaniasis in Colombia caused by Leishmania braziliensis and Leishmania panamensis. Am J Trop Med Hyg. 2012;86:807–11.

3.  Figueredo LA, de Paiva-Cavalcanti M, Almeida EL, Brandão-Filho SP, Dantas-Torres F. Clinical and hematological findings in Leishmania braziliensis-infected dogs from Pernambuco, Brazil. Rev Bras Parasitol Vet. 2012;21:418–20.

4.  Carvalho FS, Wenceslau AA, Albuquerque GR, Munhoz AD, Gross E, Carneiro PL, Oliveira HC, Rocha JM, Santos IA, Rezende RP. Leishmania (Viannia) braziliensis in dogs in Brazil: epidemiology, co-infection, and clinical aspects. Genet Mol Res. 2015;14:12062–73.

5.  Ceccarelli M, Galluzzi L, Migliazzo A, Magnani M. Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA. PLoS One. 2014;9:e88845.

6.  Pires MQ, Madeira MF, Bittencourt VR, Pacheco RS. Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation by specific PCR and RFLP-PCR assays. Rev Soc Bras Med Trop. 2014;47:243–6.

7.  Dantas-Torres F, de Paiva-Cavalcanti M, Figueredo LA, Melo MF, da Silva FJ, da Silva AL, Almeida EL, Brandão-Filho SP. Cutaneous and visceral leishmaniasis in dogs from a rural community in northeastern Brazil. Vet Parasitol. 2010;170:313–7.

8.  Passos SR, Rodrigues Tde A, Madureira AP, Giunchetti RC, Zanini MS. Clinical treatment of cutaneous leishmaniasis in dogs with furazolidone and domperidone. Int J Antimicrob Agents. 2014;44:463–5.

9.  Trevisan DA, Lonardoni MV, Demarchi IG. Diagnostic methods to cutaneous leishmaniasis detection in domestic dogs and cats. An Bras Dermatol. 2015;90:868–72.

10. Madeira MF, de O Schubach A, Schubach TM, Pereira SA, Figueiredo FB, Baptista C, Leal CA, Melo CX, Confort EM, Marzochi MC. Post-mortem parasitological evaluation of dogs seroreactive for Leishmania from Rio de Janeiro, Brazil. Vet Parasitol. 2006;138(3–4):366–70.