title: Application of qPCR in conjunctival swab samples for the evaluation of canine leishmaniasis in borderline cases or disease relapse and correlation with clinical parameters.
abstract: BACKGROUND
  In leishmaniasis caused by Leishmania infantum, the dog acts as the main reservoir for the disease. Non-invasive sampling for Leishmania detection is pivotal for rapid and affordable diagnosis. Recently, the use of conjunctival swab (CS) has been evaluated as a non-invasive sampling technique for quantitative real-time PCR (qPCR). However, few investigations have been made on the applicability of CS qPCR in particular cases such as dogs with borderline IFAT titres, suspected disease relapse with comorbidity and therapy monitoring. The aims of this study were i) to confirm the efficacy of CS, comparing these samples to buffy coat (BC) samples, as effective non-invasive samples for Leishmania quantitative detection by qPCR and ii) to verify the usefulness of qPCR compared to conventional laboratory and clinical parameters to assist in therapeutic decision making regarding dogs with complex clinical cases.
  
  
  METHODS
  Eighty dogs were divided into 4 groups based on their IFAT titres and clinical histories. Two qPCR assays were performed both on CS raw lysates and on purified DNA from BC samples. The assays were then compared. Z tests for difference of proportion, with Bonferroni correction, were carried out to evaluate the qPCR results. Logistic regression with backward stepwise elimination was performed to detect the subset of haematochemical variables significantly associated with PCR positivity.
  
  
  RESULTS
  The qPCR performed on CS samples showed better sensitivity (87%) and specificity (96%) than assays carried out using BC samples, regardless of the primers used. The haematochemical parameters haemoglobin and globulins were found to be significantly associated with qPCR positivity. Pearson correlations between Leishmania kDNA load in CS and body condition scores or IFAT titres were calculated in dogs with new leishmaniasis diagnoses. The Leishmania kDNA load in CS correlated moderately with IFAT titres (R = 0.59) but a very weak correlation (R = 0.37) with body condition score (BCS) was found.
  
  
  CONCLUSIONS
  The applicability of CS for Leishmania detection in dogs was confirmed, revealing the usefulness of raw lysates for quantitative purposes. Moreover, the qPCR was found to be particularly useful in cases lacking a clear clinical diagnosis, where the haematochemical values cannot be predictive.
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", via Saffi 2, 61029, Urbino, PU, Italy. marcelloceccarelli72@gmail.com.
authors.forename: Marcello
authors.initials: M
authors.lastname: Ceccarelli
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", via Saffi 2, 61029, Urbino, PU, Italy. luca.galluzzi@uniurb.it.
authors.forename: Luca
authors.initials: L
authors.lastname: Galluzzi
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", via Saffi 2, 61029, Urbino, PU, Italy. divide.sisti@uniurb.it.
authors.forename: Davide
authors.initials: D
authors.lastname: Sisti
authors.affiliation: Veterinary Clinic "Santa Teresa", via Piave 23, 61032, Fano, PU, Italy. barbara.bianchi@yahoo.it.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bianchi
authors.affiliation: Department of Biomolecular Sciences, University of Urbino "Carlo Bo", via Saffi 2, 61029, Urbino, PU, Italy. mauro.magnani@uniurb.it.
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000627:therapeutic use; D003228:Conjunctiva / Q000469:parasitology*; D004283:Dog Diseases / Q000175:diagnosis / Q000469:parasitology*; D004285:Dogs; D005260:Female; D007898:Leishmaniasis, Visceral / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012008:Recurrence
chemical_list: D000981:Antiprotozoal Agents