Polymerase chain reaction using conjunctival swab samples for detecting Leishmania DNA in dogs

The dog is the main domestic reservoir of Leishmania and font of infection for the vector, constituting an important host for the transmission of the parasite to humans. Non-invasive collection of swab samples for leishmaniasis diagnosis has been a promising alternative. This study analyzed the positivity of polymerase chain reaction (PCR) for the diagnosis of canine leishmaniasis in conjunctiva samples. DNA extraction was performed using SDS 20% and PCR was performed using 13A/13B primers that amplify 120-bp of Leishmania kDNA. Of the 77 dogs analyzed, 50 (64.93%) had ocular changes: 25 (32.47%) dogs had periocular lesion, 41 (53.25%) dogs had purulent eye discharge, and 17 (22.08%) dogs had both signals. PCR was positive in 35 dogs (45.45%), and there was no significant difference between dogs with and without ocular signals (p=0.4074). PCR positivity was significant higher in dogs without periocular injury (p=0.0018). Conjunctive PCR, a less invasive, fast, and painless collection technique, is indicated to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.


Introduction
The second parasitic disease that causes most deaths worldwide is leishmaniasis complex. It is endemic in Asia, Africa, the American, and the Mediterranean region. In the American continent, it is mainly found in the tropical and subtropical regions (Horimoto & Costa, 2009). The transmission of leishmaniasis occurs through the bite of female dipterans of the subfamily Phlebotominae.
As dogs are considered the main domestic reservoirs, a reliable, accurate, and specific diagnostic method for detecting parasites is crucial (Molina et al., 1994). Parasitic DNA can be detected in a broad range of clinical specimens, such as bone marrow, lymph node aspiration, and liver, kidney, or other viscera biopsies (Perez, 2015). However, these samples are obtained by invasive and painful procedures.
Serological tests are widely used; however, these tests have limitations in diagnosis as they lack sensitivity and can generate false results in dogs with clinical disease. The combination of enzyme-linked immunosorbent assay (ELISA) and dual path platform (DPP TM ) significantly improved the sensitivity and specificity (Gomes et al., 2008). Molecular methods such as polymerase chain reaction (PCR) are highly sensitive and specific for detecting the parasite's DNA, regardless of the disease stage (Oliveira et al., 2006). The need for effective methods capable of detecting the parasite by using material from the less invasive collections allows the animals to be diagnosed more painlessly. Thus, the current study analyzes the positivity of conjunctival swab PCR in the diagnosis of Canine Leishmaniasis (LeishCan) in seropositive dogs for visceral leishmaniasis.

Material and Methods
This study was reviewed and approved by the Ethics Committee on the Use of Animals of the Universidade Federal da Grande Dourados (protocol number 27/2016). Written informed consent was obtained from the dog owners.
Dogs diagnosed with LeishCan in serological tests (DPP TM rapid test and ELISA) were sent to the Control Center of Zoonoses (CCZ) in the city of Campo Grande/Mato Grosso do Sul state for euthanasia from May to July 2016.
The veterinarian at CCZ, before the collection of samples, performed the evaluation of clinical signals and the characterization of the animals regarding gender (male and female), age [puppies (up to one year old) and young adults (2-4 years old)], and size [small (below 40 centimeter), medium (40 to 60 centimeter), and large (over 60 centimeter)]. Silveira et al. (2018) analyzed the dogs, along with the samples collected from other animals in the same place and period and reported the data. The samples were collected from the conjunctiva of the right and left eye of each dog using a sterile cotton swab.
The DNA was obtained using SDS 20% and resuspended in 50 μL of TE buffer (10 mM TRIS; 1 mM EDTA; pH 8.0) according to Neitzke-Abreu et al. (2020). For each extraction, a positive control [dog blood without leishmaniasis plus 10 4 promastigotes Leishmania (Leishmania) infantum] and a negative amplification control (dog blood without leishmaniasis) were included.

Discussion
Although our results demonstrated no significant difference in PCR in dogs with eye signals, studies have demonstrated a significant association with the presence of clinical signs and positive PCR in conjunctiva samples in swab, blood, bone marrow and skin (Ferreira et al., 2012;Pereira et al., 2016). Dermatological changes, weight loss, lymphadenopathy, onychogryphosis and hepatosplenomegaly have been reported in all dogs with a positive result in conjunctive PCR. Ocular abnormalities in LeishCan are common (Penã et al., 2000;Brito et al., 2010;Baneth et al., 2008) and result from the immune response due to infection Leishmania, as the conjunctiva is one of the first lymphoid centers assets (Brito et al., 2010). The report on ocular parasitism and tissue tropism (Geisweid et al., 2013) highlights the importance of studies in canine conjunctiva for DNA research by Leishmania spp.
All animals had more than five clinical signals related to the development of the disease. In this study most animals had ocular signs (64.93%). Purulent eye discharge followed by the periocular injury was the most frequent finding of the study. Ocular signs, including keratoconjunctivitis, uveitis, and blepharoconjunctivitis, have been detected in dogs with Leishmania sp. (Ciaramella et al. 1997;Brito et al., 2010;Baneth et al., 2008). This indicating the possibility of parasites in this tissue.
The absence between the age and gender with the prevalence of LeishCan evidenced in our results corroborates with other results (Azevedo et al., 2008). However, the greater positivity in medium-sized animals in our results differs from the scientific literature that reports the greater susceptibility of large and giant breeds due to the greater body surface favoring the bite of sandflies (Gálvez et al., 2010).
PCR positivity was not high in this study (45.45%). We used primers for the Leishmania genus to detect any species, although cases of LeishCan caused by another species than L. infantum are rare. PCR is less sensitive than serological tests such as ELISA and Indirect Immunofluorescence Reaction (IIF) (Duthie et al., 2018), due the abundance of anti-Leishmania antibodies is greater than Leishmania genetic material. In addition, factors related to the method of collection, conservation and processing of samples can influence the positivity of PCR. In serology inconclusive cases (Santarém et al., 2020), molecular tests can assist in the diagnosis of LeishCan, complementing serological tests.
In our study, PCR was positive in 42.0% of dogs had ocular signals. One study showed positive conjunctival cytology for Leishmania even with negative direct parasitological examination at on lymph node and marrow samples (Santos et al., 2017). Studies have reported high positivity in the conjunctiva PCR in symptomatic animals (Ferreira et al., 2012;Pereira et al., 2016). Conjunctiva cytological investigations for amastigote research on Leishmania also demonstrated good positivity (Barbosa et al., 2012). The significant positivity in dogs without periocular injury can be explained by recent infection and the immune response. Ferreira et al. (2013) found DNA L. infantum in dogs that did not present conjunctival lesions, emphasizing the practicality and potential for diagnosis. These data indicate a significant relevance of the presence of the parasite in conjunctiva, even without tissue damage.
Using a part of the same dogs, PCR positivity was higher in blood (84.75%) (Silveira et al., 2018) and ear blood (70.90%) (Neitzke-Abreu et al., 2020) than the conjunctiva swab (45.45%), but this, in addition to being a less invasive collection allows us to obtain purer DNA and less discomfort for the animal. Other authors have shown that PCR is superior to blood (Lombardo et al., 2012;Pereira et al., 2016). The comparison in nasal and conjunctival swabs showed no difference in terms of parasitic load and was close to detection in skin biopsy, but lower than bone marrow samples (Ferreira et al., 2013). This maybe is due to a positive synergy between the DNA extraction technique and the primers used.
PCR was negative in 58.0% (29/50) dogs had ocular signals, and in 48.15 % (13/27) dogs had no ocular signals. This can indicate decreased parasitic load but show the limitation in using the conjunctiva for the diagnosis of LeishCan. Adequate cellular immune response in dogs is considered to control the spread of the pathogen in tissues such as mucous membranes and skin (Solano-Gallego et al., 2001). Ocular changes can occur due to the immune response, without the local presence of parasites. This shows the need for further studies to improve the diagnosis and understanding of ocular changes in dogs produced by Leishmania.
Although some authors affirm the significant existence of parasitic load in the ocular conjunctiva, our results did not show a good result of PCR in conjunctiva swab. The importance of exploring a less invasive, quick, and painless collection technique, in contrast to the methods for collecting samples from bone marrow and lymph nodes, helps reduce the suffering of symptomatic dogs in a weak and extremely fragile state. Conjunctive PCR is indicated in to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.