## 4 Discussion  
Although AK is less common than other forms of infectious keratitis, its incidence has increased in recent decades, as reported in a study conducted between 2009 and 2015 on 224 patients diagnosed with _Acanthamoeba_ keratitis in the Netherlands [30]. AK represents approximately 2% of all corneal infections worldwide [31], and contact lens use is one of the most common risk factors, particularly in developed countries, affecting 1-33 users per million each year [32]. The type of contact lens used also plays a role, with a more than three-fold increased risk for daily reusable lens users versus daily disposable lens users [33]. Daily reusable lens solutions can become contaminated at the point of use through contact with water or dirty hands [34], becoming a risk factor related to maintaining the sterility of the solution. Non-lens users can also develop this condition if exposed to environmental risks like contaminated tap water, swimming pools, hot tubs, and soil or dust [35; 36]. As for the therapies currently used for the treatment of infections caused by _Acanthamoeba_, these still pose important challenges, such as toxicity to human cells and resistance to the drugs used. _A. polyphaga_ is an opportunistic protozoan pathogen that is very difficult to eradicate. Therapy must be started as quickly as possible because if the parasite reaches the corneal stroma the therapeutic approach becomes more difficult, partly due to the ability of the protozoan to encryst. Cysts are in fact much more resistant to pharmacological treatment than trophozoites and require longer therapy cycles with sometimes uncertain outcomes. Therapy can, therefore, become long and demanding, and its management requires great experience because it is not always easy to evaluate the response to treatment, and complications can be very serious and difficult to manage. Resistance to therapy can develop during treatment, as well as drug-induced toxicity, with the latter leading to an initial worsening of both the inflammatory response and symptoms. New generations of drugs must be developed to help treat acanthamoebaisis, in order to  
Figure 4: The rapid destruction of _A. polyphuga_ trophozoites and the morphological changes in the residual cells observed by an inverted microscope (**a**) in the control and (**b**) after 1 h of contact with TTO/EEO at the 1 xMIC concentration. reduce both the recurrence of infection and the adverse reactions caused by the current therapies. Natural compounds like plant extracts and bacterial metabolites are very interesting and promising sources of future drugs [37; 38; 39; 40]. [PERSON] et al. [41] showed that extracts from _Passiflora_ spp. have amoebostatic and amoebicidal properties in concentrations from 4 to 12 mg/mL. A preliminary investigation into _Eryngium alpinum_ L. extracts revealed remarkable amoebicidal action against trophozoites, which reached the highest antiamoebicidal effect after two days of treatment at the concentrations of 5 mg/mL, 2.5 mg/mL, and 0.5 mg/mL [42]. In recent years, various researchers have investigated the effectiveness of nanoparticle-conjugated drugs and/or naturally occurring plant compounds against _Acanthamoeba_. In addition to significant growth inhibition, these natural compounds and nanoconjugates do not exhibit in vitro cytotoxic effects against human cells [43]. The combined use of several natural compounds allows the reduction of their concentrations and, consequently, the decrease of toxic effects during therapy [44; 45]. Other applied nanotechnologies have been shown to enhance the anti-_Acanthamoeba_ activity in the encapsulated nanoparticles, opening the way for new therapeutic options [46; 47]. Amoebicidal activity has been confirmed for the natural compounds used in the present investigation, which show remarkable effectiveness when combined. The TTO/EEO association clearly indicated a synergistic effect in all tests, and at 24 h post-treatment, no viable _A. polyphaga_ cells were observed for the 2 xMIC concentration.