Serological survey and comparison of two polymerase chain reaction (PCR) assays with enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine visceral leishmaniasis in dogs

Abstract

Visceral leishmaniasis (VL) is systemic zoonotic parasitic infection that is a health problem in some tropical and subtropical countries. The purpose of our study is to determine the seroprevalence of canine visceral leishmaniasis (CVL) in owned dogs of the Sarab area and to identify the species of Leishmania isolated from these dogs. We also compared the sensitivities and specificities of two polymerase chain reaction (PCR) assays (kDNA and ITS1) used for Leishmania infantum identification with culture, microscopic detection and enzyme-linked immunosorbent assay (ELISA) methods as well as validate the PCR techniques for the molecular diagnosis of CVL. Sera from 384 dogs of 30 villages around Sarab, were tested by ELISA and buffy coat blood fractions after sampling tested with PCR by specific primers (kDNA, ITS-18sRNA). Thirty-five dogs were seropositive by ELISA. The seroprevalence rate (SPR) of CVL was 9.1% (CI, 95% 6.6 -12.4). The most important serological result was a high proportion of seropositivity for leishmaniasis. Out of 361 (94%) asymptomatic dogs, 31 (8.6%) were seropositive, and out of 23 (6%) symptomatic dogs, 4 (17.4%) were seropositive. Agreement between
the ELISA test and clinical signs was 86.7%. Each assay was performed on 60 blood samples. PCR of kDNA (7/60 positives, 11.8%) was the most sensitive of the assays examined, followed by ELISA (3/60, 5%) and ITS1-PCR (2/60, 3.4 %). All diagnostic assays were highly specific (100 %) and had positive predictive values (PPV) >90% and negative predictive values (NPV) >88% for CVL. As expected, kDNAPCR proved to be the most sensitive (87.5 %) assay for leishmanial DNA in peripheral blood. This study shows that kDNA-PCR is significantly more sensitive than the other parasitological and serological methods, allowing the identification of infected dogs even before the appearance of serum L. infantum antibodies. Because kDNA-PCR is the most reliable, sensitive, and also a rapid diagnostic assay for CVL, it should be employed as the new standard for routine diagnosis.

Key words: Leishmania infantum, polymerase chain reaction, kinetoplast DNA, enzyme-linked immunosorbent assay, Visceral leishmaniasis, dogs, prevalence.