Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Abstract

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.

Quantification of Leishmania infantumDNA in the bone marrow, lymph node and spleen of dogs

“…bone marrow, lymph nodes and spleen) makes them important for detecting this protozoon. Different studies have demonstrated high positive rates of L. infantum detection in lymph nodes (IKONOMOPOULOS et al, 2003), spleen (SOLCÀ et al, 2012) and bone marrow (FERREIRA et al, 2012) using molecular biology tests. Some of them have suggested that lymph nodes are the best biological samples for molecular diagnosis of L. infantum (IKONOMOPOULOS et al, 2003;RAMOS et al, 2012).…”

“…Real-time PCR (qPCR) has presented high sensitivity and specificity for diagnosing and monitoring parasite loads in different biological samples (MARY et al, 2004;TUPPERWAR et al, 2008;RAMOS et al, 2012). However, based on the parasite load, it is still unclear which biological sample should be preferred in order to make a correct diagnosis (IKONOMOPOULOS et al, 2003;BARROUIN-MELO et al, 2006;SOLCÀ et al, 2012).…”

Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers

“…Bigeli et al (2012) blood. Leishmania parasites have a high number of kDNA minicircles copies (10,000 copies) per parasite, which increases the sensitivity of the assay (Quaresma et al 2009;Solc a et al 2012), as observed in this study with »10 0 parasite mL ¡1 .…”

“…Recently, PCR has significantly advanced with the use of quantitative real-time PCR (qPCR) that promotes a real-time and accurate quantification (Ramos et al 2012;Solc a et al 2012), and helps in confirmation of inconclusive cases of CVL, i.e. dogs not yet seroconverted, accurate detection of potential reservoirs, and determination of a decrease of the parasite load in these animals as a result of successful therapy.…”

Patologias genitais associadas à leishmaniose visceral canina

“…Sendo assim, o diagnóstico laboratorial pode ser realizado através do exame parasitológico direto, testes imunológicos, como imunofluorescência indireta (RIFI) ou ensaio de imunoadsorção enzimático (ELISA), métodos imunohistoquímicos e, mais recentemente, através de métodos moleculares de amplificação do ácido nucleico, como a Reação da Cadeia em Polimerase (PCR) e PCR quantitativo (Real Time PCR) (MOREIRA et al, 2007;QUEIROZ et al, 2010;SOLCÁ et al, 2012).…”