Evaluation of multiplex PCR assay for detection of Babesia spp, Ehrlichia canis and Trypanosoma evansi in dogs

A multiplex PCR test was evaluated to detect the DNA of three important dog haemoparasites by comparing with singular PCR counterpart on clinical blood samples of dogs in and around Chennai, Tamil Nadu, India. Initial screening of samples was done by microscopic examination of peripheral blood smear and singular PCR and those found exclusively positive for Babesia spp, Ehrlichia canis and Trypanosoma evansi and concurrent infections were used to standardize multiplex PCR. Amplicons of 619 bp, 377 bp and 227 bp corresponding to Babesia spp (18S rRNA gene), E. canis (VirB9 gene), and T.evansi (VSG gene) respectively were amplified, without any nonspecific amplification. The laboratory sensitivity (91.7% to 100%) and specificity (100%) of the multiplex PCR were calculated using 'true positive' and 'true negative' dog blood samples obtained in the initial screening process. Clinical blood samples from 287 dogs were screened using singular PCR and multiplex PCR tests for the presence of genome of Babesia spp, E. cards and T. evansi. The multiplex PCR was found to have high level of diagnostic specificity (97.5%-100%) in the detection of all three dog blood parasites and high level of diagnostic sensitivity (95%) in the detection of T. evansi from field level clinical blood samples compared to the singular PCR. However, the diagnostic sensitivity of the multiplex PCR was found to be low to moderate (40.45%-66.7%) in detection of Babesia spp and E. canis from field level clinical blood samples. The strength of agreement between singular and multiplex PCR assays was 'moderate' (0.445), 'good' (0.708) and 'very good' (0.968) in detection of DNA of Babesia spp, E. canis and T. evansi. The multiplex PCR was found to be 10 fold less sensitive in comparison with the singular PCR counterpart.