# A New In Vitro Model to Evaluate Anti-Adhesive Effect against Fungal Nail Infections
## 3 Results and Discussion
### In Vitro Evaluation of Fungal Adhesion to the Nail Plate
#### 3.2.2 Microbiological Assessment  
In order to define the correct testing step for evaluating the effectiveness and mode of action of different products, two different procedures for microbiological assessment were carried out. The results are presented separately below according to the procedure used. Figure 5: Contact angle measurements of the base coat treatment with respect to the membrane alone. The results obtained from the antifungal product (AP) and the base coat (BC) in the multi-treatment procedure against _Candida albicans_ and _Trichophytton rubrum_ in Figure 6A,B, respectively. In both groups, the membranes were treated three times and washed both between applications and at the end of the study before microbiological assessment. The control group membranes only underwent the washing procedure. The antiadhesive effect (AE) was expressed as the quotient of the difference of the logarithms of the colony-forming units (CFU) between the control membrane suspension (sample 3) and the colony-forming units (CFU) of the treated sample suspension in each sampling time. The higher the AE was, the higher the effectiveness of the product against fungal adhesion was. Following the international standards [33], the minimum logarithmic reduction acceptable to indicate suitable prevention of surface contamination was two. Figure 6: Antiadhesive effect of the antifungal product (AP) in group 1 and the base coat (BC) in group 2 for the multi-treatment procedure against (**A**) _C. albicans_ and (**B**) _T. rubrum_. Statistical significance: \({}^{\star}\), \(p<0.05\) is significant; \({}^{\star\star}\), \(p<0.01\) is strongly significant; \({}^{\star\star\star}\)\(p<0.001\) is very strongly significant. Thus, despite the fact that no study concerning the possible effect on nail fungal contamination has been published yet, in this work, an AE value of two or higher was chosen as the cut-off value to consider the product able to prevent fungal infection. Starting from this data, the AP seemed to be able to prevent fungal infection, particularly against _Trichophyton rubrum_. In fact, as is shown in the graphs in Figure 6, at each time, the antiadhesive activity of the AP against _Trichophyton rubrum_ was nearly double that against _Candida albicans_; specifically, after 5 h, the activity was 3.92 \(\pm\) 0.76 and 1.60 \(\pm\) 0.28, respectively. This result agrees with the literature, in which the efficacy of amorolfine evaluated by its minimum inhibitory concentration (MIC) values, which represent the lower concentration that prevents microbial growth, was reported to be stronger against _T. rubrum_ than against _C. albicans_[26; 34; 35]. The statistical analyses carried out on each group by applying a one-way ANOVA test revealed no statistical differences over time. On the contrary, significant statistical differences were obtained using a \(t\)-test between group 1 and 2 at each time against _T. rubrum_ infection. The results of the statistical analysis are evident in Figure 6B. In addition, these results, obtained after three treatments with drugs and on washed membranes, demonstrated clearly that the drug was able to penetrate deeper into the membranes to exert its action, both after washing and after only 15 min of contact. On the other hand, the AE values obtained after treatment with a water-repellent base coat showed no antiadhesive effect, since they were much lower and not significantly different at each testing time and for both fungal species. This result highlighted that a simple physical barrier cannot exert a protective effect against fungal infection. This procedure aimed to highlight the effectiveness of the product after just a single application. Figure 7 shows the results obtained from the antifungal product (AP) and the base coat (BC) in the single-treatment procedure for group 4 and 5, respectively, in which the membranes were treated once with the products and not washed. This way, the product remained on the membrane surface before microbiological assessment. Figure 7: Cont.