Avaliação do potencial de amostras clínicas de coleta não invasiva para o diagnóstico molecular da leishmaniose visceral canina por PCR

Abstract

An accurate diagnosis of canine visceral leishmaniasis (CVL) based on simple procedures for sample collection is very important as an auxiliary measure control of this severe disease. In this work we used conventional PCR to evaluate the potential of clinical samples obtained noninvasively as follows: conjunctival, nasal, oral and ear swabs for the diagnosis of CVL. In addition, we analyzed the parasite loads obtained from these samples and the possible implications using the real time PCR (qPCR). Naturally infected dogs were collected in the Municipal Zoonotic Diseases Control Department of Belo Horizonte-MG. Ten healthy no infected dogs were adopted as control group. The animals were collected in three different moments. Firstly, 80 dogs were obtained and divided into two groups with 40 dogs: group 1 without clinical signs and group 2 with clinical signs. From all these animals, the conjunctival swab from each eye, bone marrow, skin biopsy and peripheral blood were collected and submitted to the PCR-hybridization and qPCR. 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%. The PCR-hibridization positivity for conjunctival swab was equivalent (p>0.05) or higher than those for other samples (p<0.05). The qPCR data revealed that the parasite burden in the skin was the highest within each group (p<0.05), but there was no statistical difference between them (p>0.05). The antiLeishmania IgG titers from dogs with clinical manifestations were higher than those from dogs without clinical signs (p = 0.025). Positive and significant correlations were detected between parasitism and IgG titers only in the bone marrow and skin from dogs without clinical signs (p<0.05). The second and third collection enrolled 34 and 28 naturally infected dogs respectively. The same clinical samples mentioned above were collected. Additionally, the nasal swab was obtained (n = 62). The qualitative diagnosis was carried out with all these samples, except peripheral blood, using PCR with Leishmania donovani complex specific primers (cPCR). The positive index obtained from nasal swab was 87% (54/62) and this result was equivalent to those from other samples (p>0.05). Its parasite load was statistically equivalent to the parasitism in the conjunctival swab (p>0.05) and lower than the parasite burden in the bone marrow and skin (p<0.05). IV The oral and ear swabs were collected from the last 28 dogs. The cPCR positive results were as follows: oral swab, 79% (22/28); ear swab, 43% (12/28). There was statistical difference between these data (p = 0.013) and the positivity obtained from oral swab was equivalent to those from other samples (p>0.05). The parasite burden in the oral swab was equivalent to the parasitism in the conjunctival and nasal swabs (p>0.05) and lower than those estimated in the other samples (p<0.05). It was no possible to assess the parasite load in the ear swab with the qPCR. In conclusion, the clinical samples obtained noninvasively, except the ear swab, showed an important potential for the molecular diagnosis of CVL because they had a superior or equivalent performance when compared with bone marrow and skin biopsy. A special emphasis is given to the high sensitivity of PCR-hybridization with the conjunctival swab sample for detecting the infection in dogs without clinical signs. The high parasite loads in the skin of dogs with and without clinical signs represent a serious challenge for the CVL control in endemic regions, especially due to the drawbacks to detect the infection in dogs without clinical manifestations.