**3. Conclusions**

The work of Ferreira et al. (2012)[25] corroborated the conjunctival swab applicability for canine visceral leishmaniasis diagnosis. In this study the kDNA PCR-hybridization and the quantitative real-time PCR were used, respectively, for diagnosis and assessment of parasite load in clinical samples of 80 naturally infected dogs. The dogs were divided in two groups: without clinical manifestations (1) and presenting clinic signs associated with visceral leish‐ maniasis (2). All animals had positive ELISA and IFAT and/or parasitological positive test. The negative control group included 10 health dogs that tested negative in the serological and parasitological tests. The kDNA PCR-hybridization positive results rates for the clinical samples in the Group 1 were as follow: right conjunctiva, 77.5% (31/40); left conjunctiva, 75.0% (30/40); skin, 45.0% (18/40); bone marrow, 50.0% (20/40) and blood, 27.5% (11/40). By combining the results of both conjunctivas the positivity was 87.5% (35/40). For the group 2 the PCRhybridization allowed the following results: right conjunctiva, 95% (38/40); left conjunctiva 87.5% (35/40); bone marrow, 77.5% (31/40) and blood 22.5% (9/40). A positivity of 95.0% (38/40) was obtained considering the positive results of both conjunctivas. For qualitative molecular diagnosis the conjunctival swab samples showed the best results for both dogs groups. The quantitative real-time PCR was performed using primers addressed to a fragment of a singlecopy-number *L. infantum* DNA polymerase gene. Canine housekeeping β-actin gene was used as endogenous control. The results were defined as the number of parasites per 104 canine cells. For both groups the parasite burdens determinate by the quantitative real time PCR in conjunctival swab and bone marrow were statistically equivalent, by the other side the parasite load in the skin was higher than the other clinical samples. When compared between groups the parasite load from conjunctival swab in group 2 was higher than in group 1. The same relationship was found for bone marrow. However, no differences were observed in skin load between groups. The high parasite burdens detected in skin from both symptomatic and asymptomatic animals emphasized the role of infected dogs, especially the asymptomatic, as reservoir. The article considered the conjunctival swab sampling procedure suitable form molecular diagnosis of canine visceral leishmaniasis and suggested their widespread use.

208 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

An interesting study to evaluate the conjunctical swab diagnostic performance in different stages of infection and also for the follow up of dogs undergoing antileishmanial treatment was conducted by Di Muccio et al. (2012)[59]. To achieve the first objective 253 dogs from areas of endemicity from central Italy were submitted to a cross-sectional survey. For the second aim was performed a longitudinal study using 20 sick dogs under treatment. The molecular assay was a nested PCR using primes addressed to the small-subunit rRNA gene. Among the 253 animals the rates of *Leishmania* infection were 21.73% for conjunctival swab PCR, 21.34% for IFAT, 14.22% for popliteal lymph node cytological examination and 8.69% for buffy coat PCR. Seventy two dogs were positive by at least one test and considered positives for canine visceral leishmaniasis. Among these 72 dogs 76.38% were positive for conjunctival swab PCR, 75.0% for IFAT, 50.0% for lymph node cytological examination and 30.55% for buffy coat PCR. The conjunctival swab PCR showed the best performance and presented a high concordance in relation to IFAT (κ = 0.75). Test correlation with infection and clinical staging were analyzed in 54 IFAT seropositive dogs. Seven dogs were classified as exposed (low IFAT titer plus negative cytology and negative PCR), 38 as infected (low IFAT titer plus positive cytology and/ or positive PCR but without clinical signs) and 9 as sick (high IFAT titer plus positive cytology

In the Mediterranean, Southern Europe and South and Central America, with approximately 500, 000 new human visceral leishmaniasis cases reported annually and millions of dogs infected, being dogs considered to be the major reservoirs for the disease, the accurate diagnosis in these animals is extremely important. Diagnosis of canine visceral leishmaniasis is performed mainly by direct parasitological methods that can yield false-negative results, either because of the very low number of *Leishmania* spp. organisms in clinical samples (bone marrow and lymph nodes) or because morphological identification is difficult. In addition, these methods are invasive. Another problem mentioned is that serology is not sufficient as a criterion for eliminating infected dogs. Conventional serological techniques are limited by cross-reactivity with other parasitic diseases, because several technical procedures have not been standardised and due the low sensitivity of the available serological methods in the initial stages of infections.

In dogs PCR-based assay is currently the more sensitive and specific technique for detection of *Leishmania* and it allows using different clinical samples. The conjunctival swab is a noninvasive sample recently reported and up to moment few studies have been performed using this approach. Nevertheless, its high sensitivity and applicability for molecular diagnosis of canine visceral leishmaniasis have been confirmed, independently, by different research groups.

The studies demonstrated that the method allows the identification of infected dogs before the seroconversion and that conjunctival swab sensitivity for molecular diagnosis was superior or equivalent to obtained by invasive samples of either symptomatic or asymptomatic animals. The conjunctival swab was also proved useful to monitor the dogs during drug therapy. The molecular diagnosis using non-invasive samples such conjunctival swab is of great relevance in epidemiological studies when large numbers of dogs are sampled and also for clinical or experimental purposes, that implies repeated samplings. The standardization of this sampling procedure can help to become viable and widespread the molecular diagnosis of canine visceral leishmaniasis. The DNA extraction protocol and the sensitivity of PCR assay used are important variables to be considered in order to obtain the best results. Field studies in wide heterogeneous populations including seronegative and seropositive animals and works that follow up PCR positive seronegative dogs are still lacking and are very important for the method validation.

Molecular tests are yet comparatively expensive in relation to other diagnostic techniques available and require technological expertise, but considering the data presented above, a sensitive, specific and practical test could provide very cost-effective alternatives to currently available diagnostic tests, especially when used in mass-screening surveys.
