PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples

Abstract

The efficacy of conjunctival swab (CS) as a sampling method for visceral leishmaniasis (VL) diagnosis by PCR of asymptomatic dogs was evaluated. The CS was compared to blood samples (B) and skin biopsies (SB), two less invasive samples potentially useful for massive screening of dogs. Thirty asymptomatic dogs, with serological and parasitological positive tests, were used. The samples were analyzed by two PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The DNA sample volume used was of 1.0 microL and 10.0 microL respectively. Using CS samples the kDNA PCR-hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR-hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. The kDNA PCR-hybridization and ITS-1 nPCR methods showed similar sensitivities for CS and SB samples. On the other hand, for blood samples, the positivity of ITS-1 nPCR was significantly higher than the one obtained by the kDNA PCR-hybridization, indicating that sensitivity of PCR methods can vary according to the biological sample examined. Our results showed that CS was suitable to detect Leishmania DNA in asymptomatic animals when comparing to other low-invasive samples. The CS sensitivities obtained in this study were similar to the ones observed in other studies for VL diagnosis in symptomatic dogs. We concluded that the use of CS for regular screenings of dogs by PCR should be considered.

Detection of canine visceral leishmaniasis by conjunctival swab PCR

“…However, collection of such specimens is invasive, and requires skilled labor or appropriate facilities. In contrast, conjunctival swabs are fast and easy to collect (2) (3) . Thus, we assessed the suitability of conjunctival swabs as a biological sample for PCR-based diagnosis of canine visceral leishmaniasis.…”

“…For instance, previous studies demonstrated sensitivity as high as 92% (5) , and 91.7% (10) . In addition, 90% sensitivity was achieved by hybridization, and 83.3% by nested PCR (2) . L. infantum DNA was detected in conjunctival swabs from 95% of symptomatic dogs and 77.5% of asymptomatic dogs that also tested positive on other serological and parasitological tests (3) .…”

“…Notably, statistical analysis (Table 3) indicated that a positive result on conjunctival swab PCR is significantly associated with the presence of clinical signs (p < 0.05), while a positive result on blood PCR is signifi cantly associated with the absence of clinical signs (p < 0.05). Indeed, conjunctival swab PCR has been demonstrated to have high sensitivity for Leishmania in clinically diseased (12) (13) , and asymptomatic dogs (2) . We note, however, that higher sensitivity was achieved in asymptomatic dogs by other groups (2) .…”

Detection of Leishmania infantum in the smegma of infected dogs

“…In this way, the possibility of smegma contamination by contaminated semen is despised, which suggests the passage of Leishmania forms from tissue to the preputial/penile surface. The identification of L. infantum by PCR on conjunctival swab samples from infected dogs is related (Leite et al, 2010), which shows the passage of the parasite from tissue to the mucosae surface in the eye. Diniz et al (2005) found high percentage of amastigote forms of L. infantum on explants of the glans penis and preputial sheath of infected dogs (75% and 80-95%, respectively), but its presence on the smegma was not investigated.…”

Diagnóstico molecular e frequência de anticorpos anti-Leishmania infantum chagasi em cães do município de Belém, Pará

“…A padronização da PCR realizada a partir de amostras de sangue total de dois cães sintomá-ticos, positivos no exame parasitológico e reagentes na sorologia, detectou DNA de L. infantum chagasi até a diluição do DNA de 1:10, demostrando uma ótima capacidade diagnóstica em amostras de sangue total de cães sintomáticos, corroborando com os dados obtidos por outros autores (Lachaud et al 2002, Gomes et al 2007, Quaresma et al 2009, Leite et al 2010.…”