Canine visceral leishmaniasis: A comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection

Abstract

Canine visceral leishmaniasis (CVL) is endemic in northwestern Iran. This study aimed to compare real-time PCR, conventional PCR, and the direct agglutination test (DAT) for the diagnosis Leishmania infantum infection in 167 serum samples of domestic dog. Bone marrow was used for parasitological examination (smears and/or culture) in symptomatic visceral leishmaniasis, and serum was used for detection of L. infantum kinetoplast DNA (kDNA) by both conventional PCR and real-time PCR, while anti-L. infantum antibodies in sera were measured by DAT. The sera were collected from 37 symptomatic and 112 asymptomatic dogs during April to May 2011. Eighteen presumed negative samples were obtained from healthy dogs kept in non-endemic areas with no history of CVL and used as controls. All 18 samples were negative by DAT and Dipstick rK39. DAT confirmed previous exposure to L. infantum for all 149 serum samples collected from symptomatic and asymptomatic dogs in CVL endemic areas of Iran. Among the 37 symptomatic dogs, 20 (54%), 25 (67.6%), 36 (97.3%), and 37 (100%) showed L. infantum infection by parasitological methods, conventional PCR, real-time PCR, and DAT (≥1:80), respectively. Of 112 asymptomatic dogs, 79 (70.5%), 111 (99.1%), and 112 (100%) were shown to be positive by conventional PCR, and DAT (≥1:80), respectively. For ethical reasons, no asymptomatic or healthy control dogs were examined by parasitological methods. Three (16.7%) control dogs were positive by real-time PCR, but were negative by DAT, dipstick rK39, and conventional PCR methods. Parasitemia levels were measured by real-time PCR targeting kDNA using SYBR^® green assay. This quantitative technique detected infection in 89.9% (150/167) of the domestic dogs that harbored L. infantum kDNA, ranging from 0.01 49 to 310.1 parasites/ml. The average was 16.60 parasites/ml. A good agreement (0.97) was found between real-time PCR and DAT at ≥1:80 titer, used as cut-off value by Kappa analysis. Thus, real-time PCR as a quantitative PCR assay on serum samples represents a valuable tool for initial diagnosis of CVL when whole blood is not available.

Introduction

Canine visceral leishmaniasis (CVL) caused by Leishmania infantum is a zoonotic disease of the Old and the New Worlds (Gramiccia and Gradoni, 2005), and domestic dogs are considered the main animal reservoir hosts of the disease (Moreno and Alvar, 2002, Desjeux, 2004). The seroprevalence of CVL in areas of endemicity in the Mediterranean Basin and Middle East, including Iran, has been reported to be 10-37% (Fisa et al., 1999, Sideris et al., 1999, Mohebali et al., 2005, Mohebali et al., 2011). Most infected dogs do not present clinical signs, but they are seropositive particularly in endemic areas of CVL in the World (Mancianti et al., 1986, Cabral et al., 1998, Sideris et al., 1999, Moreno and Alvar, 2002, Moshfe et al., 2009).

The diagnosis methods for leishmaniasis, i.e., microscopically examination and culture as traditional tests, have limitations (Deniau et al., 2003). Direct smear examination requires expertise and lacks sensitivity (Bensoussan et al., 2006, Profeta et al., 2009). Culture is slow and labor-intensive, so results may not be available for several weeks. Serological tests such as the indirect immunofluorescence assay test (IFAT) is a routine method of diagnosing CVL, although asymptomatic dogs may yield false negative results (Almeida et al., 2005), and cross-reaction with other parasites and other Leishmania species can occur (Schulz et al., 2003, Ferreira et al., 2007). The direct agglutination test (DAT) is used as a serodiagnostic tool, because it is an appropriate and cost-effective test that does not require specialized equipment (Harith et al., 1989, Ozbel et al., 2000, Mohebali et al., 2005, Mohebali et al., 2006). Nevertheless, it has limited specificity in asymptomatic subjects, failing to distinguish between past and present

Infections due to the persistence of antibodies. It is also not reliable in immuno-compromised patients (Gradoni et al., 1993).

Validation of rapid tests for the detection of canine L. infantum infection has been reported various. Kind of Leishmania antigens, manufacturer^'s production, geographical location and clinical signs can be effect on the results of rapid test. Based on a study that was carried out by Mohebali et al. (2004) in VL endemic areas of Iran, rK39 dipstick (Cypress, Belgium) showed a moderate sensitivity (70.9%) and specificity (84.9%) in domestic dogs. Although the rK39 dipstick is more rapid, noninvasive, and does not require expertise or elaborate equipment, it seems rK39 dipstick is an appropriate, rapid test for screening of CVL in the endemic areas. Current diagnostic methods used for Leishmania infections, including enzyme linked immunosorbent assay (ELISA), require technical expertise and specialized laboratory equipment and can be labor-intensive (Reithinger et al., 2002, Abass et al., 2011).

Polymerase chain reaction (PCR) is a sensitive technique for parasite detection (Schönian et al., 2003, Cortes et al., 2004). Researchers have begun using PCR based methods for CVL diagnosis in various situations (Lachaud et al., 2001, Lachaud et al., 2002, Gomes et al., 2007). PCR assays routinely used for the diagnosis of Leishmania infection have been published (Mathis and Deplazes, 1995, Costa et al., 1996, Piarroux et al., 1996). However, the multiple steps of post-PCR manipulation require time and pose the risk of DNA contamination. The use of real-time PCR was introduced recently to detect, and/or differentiate Leishmania organisms (Schulz et al., 2003, Mary et al., 2004, Rolao et al., 2004, Van der Meide et al., 2005, Francino et al., 2006). With real-time PCR technology, monitoring of amplification of specific DNA sequences is conducted as the reaction proceeds. The method has the benefit of producing results rapidly and reducing risk of sample contamination causing false positives. Molecular techniques such as conventional PCR and quantitative real-time PCR methods provide higher sensitivity than previous serological tests and represent significant progress at the screening level as well as in diagnosis at post-therapy follow up (Ashford et al., 1995, Berrahal et al., 1996, Mary et al., 2004, Francino et al., 2006, Maia and Campino, 2008). Real-time PCR assays offer many advantages in the diagnosis of CVL in endemic areas where a large segment of the canine population is exposed to the parasite, but only a small proportion develops disease (Zaffaroni et al., 1999, Solano-Gallego et al., 2001, Francino et al., 2006).

In this study we report a sensitive, real-time PCR method using sera for detection of L. infantum in symptomatic and asymptomatic domestic dogs to assess the efficacy of its routine use in CVL diagnosis.