# Diagnosis of the Multiepitope Protein rMLEEISH3 for Canine Visceral Leishmaniasis
## 3 Results and Discussion
### Diagnostic Performance of rMELEISH3 Antigen in Detecting CVL
The results of ELISA tests involving the interaction between the rMELEISH3 antigen (100 ng/well) and serum samples derived from dogs with different parasite loads, together with those from positive and negative control dogs, are shown in Figure 5. ANOVA revealed that there were significant differences between groups [F(4, 31) = 29.96; \(p<0.0001\)], indicating that the absorbance means varied significantly among the five groups tested. Because the _F value_ represents the ratio of the observed between-group variability to the within-group variability, the higher the _F value_, the stronger the evidence for true differences between groups. [PERSON]'s post hoc tests confirmed that the differences between the negative and positive control samples were statistically significant (\(p<0.05\)), as well as the differences between positive samples with high, medium and low parasite loads in the bone marrow. These results indicate that the rMELEISH3 antigen not only recognizes anti- _L. infantum antibodies_ but also discriminates between samples with different reactivities. Figure 5: Evaluation of rMELEISH3 antigen performance. Results of enzyme-linked immunosorbent assays (ELISA) performed according to an in-house protocol with an antigen concentration of 100 ng/well and serum samples derived from _L. infantum-infected dogs_ (n = 23) presenting high (n = 6), medium (n = 8), and low (n = 9) parasite loads. Positive (n = 5) and negative (n = 8) control samples were included in the assay. The dashed line indicates the cutoff point, and the bars represent the standard deviation of the measured values. Statistical analysis by one-way ANOVA revealed significant differences between groups (F(4, 31) = 29.96; \(p<0.0001\)). Abbreviations: ABS, absorbance; AUC, area under the curve; SEN, sensitivity; SPC, specificity. Antibody detection in dogs with low parasite loads poses a challenge in the diagnosis of CVL using conventional serological tests, such as DPP and ELISA, as sensitivity tends to be reduced at low loads, especially in asymptomatic animals. For example, [PERSON] et al. [11] reported that although ELISA had a sensitivity of 95.2% for symptomatic dogs, the value dropped to 65.6% for asymptomatic dogs. This limitation is associated with low antibody production during the early stages of infection (asymptomatic dogs with low parasite load), when the immune response is predominantly cellular. Furthermore, the heterogeneous distribution of the parasite in tissues may make detection difficult even when molecular techniques are employed [15; 36]. The results presented here show that the rMELEISH3 antigen presented high sensitivity, as it was able to detect antibodies in sera from dogs with low parasite loads (0.07 to 0.35 parasites/ug of tissue DNA), demonstrating the effectiveness of the assay even in cases of subclinical infection. This performance is comparable to that of the _Leishman amastigote_--specific protein rLiHyp1, which has been shown to induce a robust immune response and has been considered a potential candidate for improving the serodiagnosis of asymptomatic and symptomatic CVL, as well as for the development of a vaccine [15]. The ability of rMELEISH3 to identify infection during the early stages is particularly relevant for the control of CVL, since even asymptomatic dogs are reservoirs of the parasite and perpetuate disease transmission [37]. The ROC curve constructed for the ELISA tests using rMELEISH3 at a concentration of 100 ng per well (Figure 6) shows that the assay presented a sensitivity of 96.4%, a fundamental value that ensures the detection of true positive samples, and a specificity of 100%, ensuring the correct identification of true negative samples. The AUC value of 0.996 indicates a near-perfect diagnostic performance in discriminating between reactive and nonreactive sera. Figure 6: Receiver Operating Characteristic (ROC) curve related to enzyme-linked immunosorbent assays (ELISA) performed according to an in-house protocol with an antigen concentration of 100 ng/well and serum samples from _L.