Analytic performance of a rapid quantitative assay for measuring anti-Leishmania antibodies in dog serum samples

Canine leishmaniosis (CanL) is a complex disease caused by Leishmania infantum. In endemic areas the proportion of infected dogs is much higher than that of animals with CanL. Quantitative antibody (ab) measure is the first step in the diagnostic approach of suspected cases and in post-therapy follow-up [1]. Antibody detection is also required in healthy dogs in case of annual health check, blood donors, anti-Leishmania vaccination, relocation from/after travelling to endemic areas). The immunofluorescence ab test (IFAT) is a reference quantitative method for measuring anti-Leishmania abs. Qualitative serological point-of-care (POC) tests are available and their accuracy is variable, particularly in terms of sensitivity. A quantitative assessment of dog anti-Leishmania abs is obtained by a POC fluorescence immunochromatographic assay (cLSH-Ab, GUANGZHOU EXCBIO TECHNOLOGY CO., Ltd, Guangzhou, PRC) and automated reading of results [test units per milliliter (TU/mL)] by an immunofluorescence analyzer (FIAVETTM). A categorical semi-quantitative interpretation of results (negative, suspicious, weak positive, medium positive, strong positive) is provided. No published studies have validated this test under field conditions, and the aim of this pilot study was to compare the analytic performance of the cLSH-Ab immunoassay with conventional IFAT, evaluating agreement and diagnostic performance at the two IFAT cut-off dilution mostly used for diagnosing CanL (1:80 and 1:160). The sample size was calculated based on a statistical power of 95%, a type I error of 5%, and an expected disagreement rate between 20% and 28%. Agreement between the two tests was evaluated using Cohen’s kappa coefficient (κ), and diagnostic performance parameters [accuracy (Acc), sensitivity (Sen), specificity (Spe), positive predictive value (PPV), and negative predictive value (NPV)] were calculated. A total of 69 convenience serum samples of dogs from Sardinia were double-blind tested for anti-Leishmania abs with the cLSH-Ab immunoassay (according to the manufacturer’s instructions) and IFAT [2]. Leftover serum aliquots from clinically healthy dogs tested prior to L. infantum vaccination, suspected cases, and from post-therapy follow-up were used. Based on the cLSH-Ab immunoassay, 52 samples were negative and 17 tested positive (two medium positive and 15 strong positive). At the 1:80 IFAT cut-off, one false positive (medium positive) and eight false negatives (IFAT titers 1:80-1:160) were found (Acc 87.0%, Sen 66.7%, Spe 97.8%, PPV 94.1%, NPV 84.6%), and substantial agreement was seen (κ= 0.681). At the 1:160 cut-off, one false positive (medium positive) and one false negative (IFAT titer 1:160) were found (Acc 97.1%, Sen 94.1%, Spe 98.1%, PPV 94.1%, NPV 98.1%) and a strong agreement (κ= 0.922) was seen. Most discrepancies (12%) occurred at cut-off IFAT level (1:80), whereas the cLSH-Ab immunoassay reliably identified positive samples with higher titers (≥160). Further studies, including also “suspicious” and “weak positive” samples, are needed to confirm that cLSH-Ab immunoassay provides a reliable POC quantitative assessment of anti-Leishmania abs in clinical settings.