# A New In Vitro Model to Evaluate Anti-Adhesive Effect against Fungal Nail Infections
## 4 Conclusions  
The applied in vitro method for membrane contamination showed itself to be effective for the fungal species used. Thus, hood membranes were shown to be a valuable in vitro model to consider for this kind of product assessment. Hoof membranes can be produced with specific characteristics to mimic a wide range of nail conditions (e.g., healthy or dystrophic). Moreover, the selection of flaky samples through morphological assessment, as well as the inner higher permeabilities of the membranes, allow for the set-up of an efficacy test within extreme conditions highly favoring fungal contamination. However, some challenges were faced with the model, and they need to be taken into account, such as the flaking of the membrane itself, which can become more permeable in an aqueous environment. The preliminary sanitization procedure carried out on the membranes permitted the obtaining of the substrate with very low microbial contamination, and thus it was very useful for the aim of this test. 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, actually used in international guidelines for surface contamination, was chosen as the cut-off value to consider a product able to prevent fungal infection. The protocol used in this work permitted identifying a suitable negative control in a water-repellent base coat, which can be used in all further studies as a reference. The results obtained from microbiological experiments evidenced clearly that the preventing activity of a product changes dramatically in relation to fungal species. In fact, even an antifungal drug like amorolfine could exert statistically significant antiadhesion activity against only one fungus; its activity was particularly high against _T. rubrum_. \begin{table}
\begin{tabular}{c c c c c} \hline \multicolumn{5}{c}{**Multiple Comparisons Test against _Tricophyllum rubrum_**} \\ \hline
**Comparison Groups** & **Mean Difference** & **95.00\% CI of Diff.** & **Significant** & \(p\)**Value** \\ \hline
1 vs. 4 & 1.503 & \(-\)0.2805 to 3.287 & No & 0.1018 \\ \hline
1 vs. 2 & 3.440 & 1.656 to 5.224 & Yes & 0.0012 ** \\ \hline
1 vs. 5 & 4.067 & 2.283 to 5.851 & Yes & 0.0004 *** \\ \hline
4 vs. 2 & 1.937 & 0.1528 to 3.721 & Yes & 0.0341 * \\ \hline
4 vs. 5 & 2.563 & 0.7795 to 4.347 & Yes & 0.0076 ** \\ \hline
2 vs. 5 & 0.6267 & \(-\)1.157 to 2.411 & No & 0.8313 \\ \hline \multicolumn{5}{c}{**Multiple Comparisons Test against _Candidia albicans_**} \\ \hline
**Comparison Groups** & **Mean Difference** & **95.00\% CI of Diff.** & **Significant** & \(p\)**Value** \\ \hline
1 vs. 4 & \(-\)0.003333 & \(-\)4.240 to 4.233 & No & \(>\)0.9999 \\ \hline
1 vs. 2 & 1.763 & \(-\)2.473 to 6.000 & No & 0.5695 \\ \hline
1 vs. 5 & 2.883 & \(-\)1.353 to 7.120 & No & 0.2085 \\ \hline
4 vs. 2 & 1.767 & \(-\)2.470 to 6.003 & No & 0.5682 \\ \hline
4 vs. 5 & 2.887 & \(-\)1.350 to 7.123 & No & 0.2077 \\ \hline
2 vs. 5 & 1.120 & \(-\)3.117 to 5.357 & No & 0.8313 \\ \hline \end{tabular}
\end{table} 
Table 1: Statistical analysis performed on all groups by a one-way analysis of variance (ANOVA) multiple comparisons test. Statistical significance: *, \(p\) \(<\) 0.05 is significant; **, \(p\) \(<\) 0.01 is strongly significant; ***, \(p\) \(<\) 0.001 is very strongly significant. Furthermore, taking into account the results obtained at all testing times, 5 h appeared to be the best time to test antiadhesive activity. Finally, statistical analysis permitted concluding that the antiadhesive activity found between multiple treatments and one single application of an AP was the same, highlighting that the drug could penetrate into the membrane to maintain its activity against _T. rubrum_ even when washed. This work represents a starting point for the development of a new method to evaluate the protective effects of drugs, medical devices or cosmetics against fungal adhesion, using hood membranes as a model.