The use of conjunctival swab samples for PCR screening for visceral leishmaniasis in vaccinated dogs

Abstract

The polymerase chain reaction (PCR) has been shown to provide a rapid and sensitive technique for Leishmania detection. The aim of this study was to evaluate the technique of noninvasive conjunctival swabs (CS) as a sampling method for molecular screening for visceral leishmaniasis (VL) in a group of 42 police dogs, all of them vaccinated against VL, and to compare the results with those obtained by serological tests. The serological assays were performed independently by three laboratories. Laboratories 1 and 2 were private laboratories and laboratory 3 was the National Reference Laboratory. The first serological screening performed by laboratory 1 showed 15 reactive dogs and 4 indeterminate. Laboratory 2 confirmed only 3 reactive dogs and 2 indeterminate. Laboratory 3 confirmed 7 reactive dogs and 3 indeterminate. The PCR diagnosis using the CS procedure was performed on all 42 animals and was able to detect Leishmania DNA in 17 dogs. The PCR assay confirmed all the cases that were simultaneously reactive in the serological tests by two laboratories. The results showed that the CS technique was a sensitive and practical method for sample collection, thus allowing reliable diagnostic tests through PCR.Keywords: Visceral leishmaniasis, dog, diagnosis, PCR, conjunctival swab. ResumoA PCR (do inglês Polymerase Chain Reaction) tem demonstrado ser uma técnica rápida e sensível para detecção de Leishmania. O objetivo deste estudo foi avaliar a técnica não invasiva do swab conjuntival na identificação por PCR de animais infectados em um grupo de 42 cães policiais, todos vacinados contra a Leishmaniose Visceral (VL), e comparar os resultados com aqueles obtidos pelos testes sorológicos. Os ensaios sorológicos foram realizados independentemente por três laboratórios. Os laboratórios 1 e 2 eram privados. O laboratório 3 era o Laboratório de Referência Nacional. A primeira triagem sorológica realizada pelo laboratório 1 apresentou 15 cães reativos e 4 indeterminados. O laboratório 2 confirmou apenas 3 cães reativos e 2 animais indeterminados. O laboratório 3 confirmou 7 cães reativos e 3 cães foram classificados como indeterminados. O diagnóstico pela PCR, utilizando o procedimento do swab conjuntival, foi realizado em todos os 42 animais e foi capaz de detectar DNA de Leishmania em 17 cães. A PCR confirmou todos os casos simultaneamente reativos nos testes sorológicos de dois laboratórios. Os resultados demonstraram que o swab conjuntival é um método sensível e prático para coleta de amostra, permitindo um diagnóstico consistente através da PCR.

Course of experimental infection of canine leishmaniosis: Follow-up and utility of noninvasive diagnostic techniques

“…Conjunctival swabs have offered promising results for a noninvasive molecular diagnosis of CanL including a high sensitivity (over 90%) (Carvalho Ferreira et al, 2014;Strauss-Ayali et al, 2004) and specificity (100%) (StraussAyali et al, 2004) even in seronegative (Leite et al, 2011) and clinically healthy dogs (Carvalho Ferreira et al, 2014). Amastigotes can reach the ocular region via the bloodstream (Reithinger et al, 2002) and most likely also via the lymphatic tissue of the conjunctiva (Strauss-Ayali et al, 2004).…”

Molecular detection of visceral leishmaniasis in dogs from Barão de Melgaço, Pantanal region of Mato Grosso, Brazil

“…These values are higher than those reported by Almeida et al (2013) in the capital of Mato Grosso, Cuiabá -an area of high endemicity for VL, who found the parasite DNA in 4.6% (20/430) of the leukocyte covered samples analyzed. In addition, the occurrence of molecular prevalence higher than the serological findings of Dias et al ( 2017) is justified and described in the literature (Coura-Vital et al 2011, Leite et al 2011) since, due to the greater sensitivity and specificity of molecular tests, they could detect the infection before seroconversion (Strauss-Ayali et al 2004). Furthermore, Nunes et al (2015), in a longitudinal approach of a canine population, found that detection of CVL through PCR in blood samples showed greater sensitivity than that in the tests recommended by the MS in the first six months of follow-up.…”

Evaluating the specificity of primers employed for PCR-based diagnostics of Leishmaniases using multiple alignment analysis

“…The advent of the molecular diagnosis for detection of numerous diseases by using polymerase chain reactions (PCRs) has provided new strategies to minimize eventual technical limitations. Within this context, PCRs have been extensively employed as a quick, specific and sensitive technical approach for detection of Leishmania (Reithinger, Lambson, Barker, Davis, 2000;Bensoussan, Nasereddin, Jonas, Schnur, 2006;JaffeMaia & Campino, 2008;Queiroz, Assis, Oliveira, Machado, Nunes et al, 2010;Leite, Mendes, Ferreira, de Andrade, 2011). According to Ye et al (2012), specific primers are designed for detection of organismal DNA and the homology between such primers and target DNA confers high specificity to PCR-based diagnosis, since the identification of the amplification products under given reaction conditions reveals the presence of an infectious organism.…”