Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs

Abstract

The importance of dogs as a reservoir for Leishmania infantumchagasi in urban environments has stimulated numerous studies assessing diagnostic techniques. When performed properly, such procedures are an important step in preventing leishmaniasis in humans. Molecular methods have become prominent for this purpose. The aim of the present study was to determine the performance of the polymerase chain reaction (PCR) and real-time PCR (qPCR) for diagnosing of canine visceral leishmaniasis (CVL) using different biological samples. For this, 35 dogs from an area endemic for CVL were used. Bone marrow aspirate and lymph node and spleen fragments from these dogs were used for the molecular diagnosis. In the present study, qPCR was able to detect a greater number of positive animals than seen with PCR. Among the different biological samples used, there was no significant difference in L. infantumchagasi DNA detection between PCR and qPCR. However, considering that lymph nodes are easy to acquire, these can be considered to be the best samples for making molecular diagnoses of L. infantum chagasi infection.

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

“…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). Nonetheless, few studies on parasite load quantification have been conducted, even though such information is extremely important for correctly choosing the sample to be used.…”

“…In turn, obtaining lymph node aspirates not only is easier from a practical point of view but also demonstrates the greater viability of this biological sample over bone marrow and spleen samples. In addition, this biological sample (lymph node) has been demonstrated to be useful for detecting the protozoon, independently of the animal's clinical status (RAMOS et al, 2012).…”

“…Conversely, molecular methods have acquired considerable importance for diagnosing many diseases, including CVL. 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).…”

Novel markers of inflammatory response and hepatic dysfunction in canine leishmaniasis

“…They were negative for Leishmania braziliensis. According to researchers [18], L. infantum is the main agent when PCR results are positive, especially in association with geographic localization of dogs, and presence of clinical signs. In addition, sera samples from 17 healthy dogs, negative for L. infantum (serologic and PCR), composed our negative control group.…”

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

“…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.…”