Optimization and validation of a TaqMan real-time PCR for the detection of Heterobilharzia americana in dog feces

Heterobilharzia americana is a trematode parasite that causes canine schistosomiasis, a disease capable of causing significant morbidity and mortality in dogs. Reliable diagnosis is essential for limiting disease progression. This study aimed to optimize and validate a previously developed TaqMan qPCR assay for detection of H. americana DNA in feces. The assay targets a highly repetitive non-coding DNA sequence located across multiple loci within the H. americana genome. PCR optimization involved gradient thermocycling and serial dilutions to refine annealing temperature and efficiency. PCR products were cloned and sequenced to confirm target specificity. Analytical sensitivity was assessed using serial two-fold dilutions of H. americana eggs spiked into feces and matrix-free samples, with PCR detection across replicates. Diagnostic sensitivity was assessed in 111 fecal samples from infected dogs using fecal sedimentation as gold standard. Analytical and diagnostic specificities were assessed by testing 54 fecal samples containing non-target parasites and 100 fecal samples from H. americana-uninfected dogs, respectively. Target specificity was confirmed by BLAST. Assay efficiency was 107 %. Matrix-dependent analytical sensitivity was 3 eggs/g feces (100 % replicates) and 1.5 eggs/g feces (60 % replicates); matrix-free sensitivity was 3 eggs/mL in saline. Diagnostic sensitivity was 98.2 % (95 % CI: 93.7-99.8 %). Both analytical and diagnostic specificities were 100 % (95 % CI: 93.6-100 % and 96.4-100 %, respectively). The assay demonstrated low intra-assay and inter-assay variability and minimal inter-operator variability across the dilution range tested. These findings support the optimized TaqMan qPCR assay as a highly sensitive and specific tool for detecting H. americana DNA in dog feces.