Evaluation of Immunofluorescence Antibody Test Used for the Diagnosis of Canine Leishmaniasis in the Mediterranean Basin: A Systematic Review and Meta-Analysis

<div><p>With an expected sensitivity (Se) of 96% and specificity (Sp) of 98%, the immunofluorescence antibody test (IFAT) is frequently used as a reference test to validate new diagnostic methods and estimate the canine leihmaniasis (CanL) true prevalence in the Mediterranean basin. To review the diagnostic accuracy of IFAT to diagnose CanL in this area with reference to its Se and Sp and elucidate the potential causes of their variations, a systematic review was conducted (31 studies for the 26-year period). Three IFAT validation methods stood out: the classical contingency table method, methods based on statistical models and those based on experimental studies. A variation in the IFAT Se and Sp values and cut-off values was observed. For the classical validation method based on a meta-analysis, the Se of IFAT was estimated in this area as 89.86% and 31.25% in symptomatic and asymptomatic dogs, respectively. The Sp of IFAT was estimated in non-endemic and endemic areas as 98.12% and 96.57%, respectively. IFAT can be considered as a good standard test in non-endemic areas for CanL, but its accuracy declines in endemic areas due to the complexity of the disease. Indeed, the accuracy of IFAT is due to the negative results obtained in non-infected dogs from non-endemic areas and to the positive results obtained in sera of symptomatic dogs living in endemic areas. But IFAT results are not unequivocal when it comes to determining CanL infection on asymptomatic dogs living in endemic areas. Statistical methods might be a solution to overcome the lack of gold standard, to better categorize groups of animals investigated, to assess optimal cut-off values and to allow a better estimate of the true prevalence aiming information on preventive/control measures for CanL.</p></div

Statistics applied to optical densities obtained with the E^rns–based Ag ELISA on 68 field negative samples, according to the protocol (P) and day (D) used.

<p>Statistics applied to optical densities obtained with the E^rns–based Ag ELISA on 68 field negative samples, according to the protocol (P) and day (D) used.</p

Parameters that might influence IFAT sensitivity and specificity.

<p>Parameters that might influence IFAT sensitivity and specificity.</p

The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Flow Diagram.

<p>To the left are different steps of the PRISMA flow diagram; to the right are the results obtained after each of these steps.</p

Description of the protocols used in the study.

<p>Description of the protocols used in the study.</p

Kinetic of median optical density obtained with the Erns–based Ag ELISA in function of dilution of five positive reference samples at three different time periods for each of the four different protocols used.

<p>Legend: P1, P2, P3, P4: protocols applied to the samples; P1 and P3: no significant difference in function of time (two-factor ANOVA with repeated measures on one factor; P-value > 0.05); P2 and P4: significant difference in function of time (two-factor ANOVA with repeated measures on one factor; P-value < 0.05).</p

Estimated sensitivity and specificity of IFAT based on the systematic review and meta-analysis (based on available studies from classical validation method).

<p>The specificity was estimated in non-endemic and endemic area. The sensitivity was estimated in asymptomatic and symptomatic dogs in endemic Mediterranean basin. The sensitivity of the IFAT in endemic areas was estimated using a sum of 103 asymptomatic dogs (32 of them were positive in IFAT) and using a sum of 173 symptomatic dogs (161 of them were positive in IFAT). The corresponding numbers of Se studies for asymptomatic and symptomatic dogs were [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref050" target="_blank">50</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref053" target="_blank">53</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref060" target="_blank">60</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref081" target="_blank">81</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref050" target="_blank">50</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref052" target="_blank">52</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref053" target="_blank">53</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref054" target="_blank">54</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref060" target="_blank">60</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref081" target="_blank">81</a>] respectively. The corresponding numbers of Sp studies for non-endemic and endemic area were [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref053" target="_blank">53</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref054" target="_blank">54</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref060" target="_blank">60</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref076" target="_blank">76</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref048" target="_blank">48</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref054" target="_blank">54</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref055" target="_blank">55</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref057" target="_blank">57</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref058" target="_blank">58</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref059" target="_blank">59</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref076" target="_blank">76</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.ref080" target="_blank">80</a>] respectively (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161051#pone.0161051.s001" target="_blank">S1 Table</a></b>). Heterogeneity tests were significant (p = 0.01 and p<0.001, for the Se, and the Sp respectively).</p

Statistics of photometric determination of DNA concentration.

<p>Statistics of photometric determination of DNA concentration.</p

Description of the reporting system for wildlife surveillance of bovine tuberculosis in Belgium.

<p>Description of the reporting system for wildlife surveillance of bovine tuberculosis in Belgium.</p

Kinetic of median optical density obtained with the Erns–based Ag ELISA using dilution of five reference samples.

<p><b>Four different protocols were used at each of the three different time periods (day 1 in A, day 3 in B and day 14 in C).</b> Legend: P1, P2, P3, P4: protocols applied to the samples; No significant difference between groups was found at day 1 and 14 (two-factor ANOVA with repeated measures on one factor; P-value > 0.05); significant difference between groups (two-factor ANOVA with repeated measures on one factor, P-value < 0.05) was found at day 3.</p