Evaluation of conjunctival swab as a mass-screening tool for molecular diagnosis of canine visceral leishmaniasis.

The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil once the dog is the main reservoir host of the disease. The aim of this study was to evaluate the conjunctival swab (CS) as a mass-screening tool for CVL molecular diagnosis in an endemic area classified as priority for the Brazilian Ministry of Healthy for surveillance action. A total of 1350 domiciled dogs were screened. The animals were evaluated by serological tests (enzyme-linked immunosorbent assay (ELISA) as screening and immunofluorescence antibody test (IFAT) for confirmation) and by CS associated to real-time PCR, using primers addressed to kinetoplast DNA (kDNA) minicircles and SYBR Green. Canine ß-globin gene amplification was used to evaluate the sample DNA integrity. A subgroup of 484 animals was also submitted to clinical evaluation. Among the 1350 dogs screened, 369 (27.3%) were positive by CS real-time PCR and 126 (9.3%) tested positive by ELISA. Thirty-one percent (39/126) of the ELISA-positive dogs were confirmed by IFAT. CS real-time PCR was able to detect infection in dogs independently of the symptomatology degree (p > 0.05), while ELISA was more sensitive in the group of dogs that present three or more clinical signs related to CVL. The results demonstrated that CS real-time PCR was able to detect a higher number of infected dogs than ELISA and that the prevalence of canine infections has been underestimated by the serological assays. The use of sensitive molecular diagnostic methods like CS real-time PCR, mainly in endemic areas, could greatly contribute to disease control.

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

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.

The use of conjunctival swab samples for PCR screening for visceral leishmaniasis in vaccinated dogs / O uso de amostras de swab conjuntival para triagem por PCR da leishmaniose visceral em cães vacinados

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.

A 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.