Diagnostic value of digital PCR (dPCR) in the diagnosis of leishmania infantum from canine chronic dermatitis

The diagnosis of CanL can be challenging due to its broad clinical spectrum and frequent occurrence of subclinical infections in endemic areas. Molecular methods, particularly qPCR, have become pivotal in CanL diagnosis. The study aimed to compare the diagnostic performance of digital PCR (dPCR) and qPCR, which is currently considered the gold standard for the quantitative detection of L. infantum DNA. Skin biopsies from 37 dogs living in CanL-endemic areas were selected based on histopathological criteria. DNA was extracted from FFPE tissue and Leishmania detection was performed using both qPCR and dPCR with a Leishmania-specific assay. Immunohistochemistry was additionally performed on samples in which Leishmania DNA was detected, provided that sufficient material was available. Different statistical tests were used to compare the performance and agreement between dPCR and qPCR. dPCR demonstrated excellent technical performance, with an average of ∼25,000 partitions per sample, and detected significantly more copies than qPCR, especially in samples with low DNA concentrations. While quantitative agreement was substantial (Lin's CCC = 0.86), dPCR systematically measured higher values. At a detection threshold of 1 copy/μL, dPCR achieved 100% sensitivity, highlighting a superior detection capability. However, the specificity decreased to 62%, likely reflecting the detection of additional low-level positives. Cohen's κ indicated moderate categorical agreement (0.54) between the two methods. Overall, the results show that dPCR represents a novel and promising quantitative molecular technique for the detection of Leishmania DNA in FFPE skin samples.