Evaluation of hair samples for PCR detection of Leishmania infantum in dogs

Canine leishmaniosis (CanL) caused by Leishmania infantum remains a major public health concern in endemic regions, where dogs are the primary domestic reservoir. Despite high accuracy, the invasiveness of current diagnostic methods limits their practical application. This study evaluated the diagnostic potential of hair as a non-invasive sample for molecular detection of L. infantum using a nested PCR targeting the ITS1 region (HAIR-test). All PCR-based tests in this study, including those performed on lymph node aspirates (LN-test), blood (BL-test), and conjunctival swabs (CS-test), employed the same nested ITS1-PCR protocol. A cohort of 44 naturally infected dogs, defined by parasitological confirmation or LN-test positivity, was evaluated using the HAIR-test, with hair samples independently collected by two different operators. The combined sensitivity of the HAIR-test was 59.1 %, outperforming the BL-test and matching the performance of the CS-test. Sequencing confirmed L. infantum DNA in hair samples, but also revealed co-amplification of non-Leishmania trypanosomatids in a subset, underscoring the need for high-specificity assays. Inter-collector agreement was moderate, indicating potential variability in parasite distribution or inconsistencies in sample collection and handling. No correlation was observed between hair mass and PCR-positivity, suggesting that L. infantum DNA is present in follicular bulbs rather than in hair shafts. Beyond suboptimal sensitivity, limitations in assay reproducibility and uncertainties regarding the precise origin of the detected DNA underscore the need for further methodological refinement and validation. The HAIR-test offers a viable, non-invasive alternative in settings where invasive sampling is impractical, but its routine diagnostic application requires methodological refinements and validation.