Canine Skin and Conjunctival Swab Samples for the Detection and Quantification of Leishmania infantum DNA in an Endemic Urban Area in Brazil

Abstract

Background We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively. Methodology/Principal Findings Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti- Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups. Conclusions The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.

Leishmania (Viannia) braziliensis in dogs in Brazil: epidemiology, co-infection, and clinical aspects

“…The use of PCR for whole blood specimens is a practical and only mildly invasive technique that can be used in epidemiological studies involving large numbers of animals, although different tissues and secretions can be used (Silva et al, 2010;de Almeida Ferreira et al, 2012).…”

First detection of Leishmania kDNA in canine cerumen samples by qPCR

“…According with many authors, blood was the sample with the lowest sensitivity (Leite et al, 2010;De Almeida Ferreira et al, 2012;Lombardo et al, 2012): 12 dogs that were negative in blood had positive results in cerumen-qPCR and, on the contrary, only one dog was positive to blood and negative to cerumen.…”

“…Lymph node, bone marrow and skin biopsies provide high sensitivity results (Solano-Gallego et al, 2001;De Almeida Ferreira et al, 2012). Blood samples have lower sensitivity (Solano-Gallego et al, 2011), although the collection is easier (Manna et al, 2004).…”

“…However, all these samples are obtained by invasive methods that cause animal distress and owner reluctance, so it is important to test non-invasive samples as an alternative. In this way, recent evidences suggest the potential use of conjunctival, oral, nasal, ear and vulvar swabs for sampling, taking cells from the mucosa or from other anatomical regions (De Almeida Ferreira et al,2012;Ferreira et al, 2013;Hernández et al, 2015). Moreover, it has been previously reported that hair is a suitable sample for the molecular diagnosis of leishmaniosis in dogs, mice and wild mammals Iniesta et al, 2013;Muñoz-Madrid et al, 2013), as it does not cause any harm or stress to the animals, and the sensitivity of the technique is similar to that obtained using blood samples.…”

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

“…The advantages of real-time quantitative PCR (qPCR) approaches in comparison with conventional PCR assays include the potential for increased sensitivity and specificity and for a decreased risk of contamination of the laboratory environment (14,(18)(19)(20)(21)(22)(23)(24)(25)(26). The efficiency of real-time platforms for Leishmania species identification has been discussed (24,(27)(28)(29)(30).…”