ESTUDIOS CLÍNICOS

Sporicidal effect of amorolfine and other antimycotics used in the therapy of fungal nail infections.
Seidl HP, Jäckel A, Müller J, Schaller M, Borelli C, Polak A.
Mycoses. 2015 Oct;58(10):610-9. doi: 10.1111/myc.12369.

Although topical antifungal therapies for treating onychomycosis are available, the cure rate is unsatisfactorily low with a simultaneously high risk of recurrence. One reason might be the formation of dormant fungal cells by the pathogen, known as spores, which can survive in the affected nail keratin, thereby evading the effect of antifungal drugs. In this in vitro study, the ability of amorolfine and four other antimycotics (ciclopirox, bifonazole, terbinafine and fluconazole) to kill microconidia of the dermatophyte Trichophyton rubrum, chlamydospores of the dermatophyte Epidermophyton floccosum and blastospores of the yeast Candida albicans was extensively studied as these fungi occur predominantly in onychomycosis. The effectiveness of all five antimycotics depended on the drug concentration and the incubation time: a concentration of 10–1000 times the minimum inhibitory concentration against growing hyphae cells is needed to exert a sporicidal action. Amorolfine and ciclopirox showed the same sporicidal efficacy and kinetics for all three varieties of spores. Both were more effective than fluconazole and bifonazole against microconidia and chlamydospores as well as slightly more potent against chlamydospores and blastospores than terbinafine after 4 days of incubation and at concentrations of ≥10 lg ml
1. Finally, sporicidal activity on the tested strains was demonstrated for all five different antimycotics used for onychomycosis treatment.

Agar sublimation test for the in vitro determination of the antifungal activity of morpholine derivatives.
Polak A, Jäckel A, Noack A, Kappe R.
Mycoses. 2004 Jun;47(5-6):184-92. doi: 10.1111/j.1439-0507.2004.00975.x.

We studied the in vitro antifungal activities of a wide range of antimycotic agents, including amorolfine, terbinafine, naftifine, five morpholine derivatives, ciclopiroxolamine, bifonazole, clotrimazole, ketoconazole, itraconazole, fluconazole, voriconazole, flucytosine, amphotericin B, nystatin, and caspofungin, against Candida albicans and Trichophyton rubrum by conventional agar diffusion tests and by a novel sublimation method. For the sublimation method, 6 mm filter paper disks were soaked with defined amounts of antimycotic drugs, air dried, placed in the center of the lids of 9 cm Petri dishes, and incubated upside down with inoculated agar plates 10 mm above the disks. The conventional disk diffusion tests produced inhibition zones as previously described. The disk sublimation tests produced large inhibition zones with amorolfine, five amorolfine derivatives, and terbinafine, but with none of the other antifungal agents. Possible therapeutic advantages of agents, which are able to overcome air cavities in mycotic lesions, e.g. in onychomycosis, are discussed.

Efficacy of amorolfine nail lacquer for the prophylaxis of onychomycosis over 3 years.
Sigurgeirsson B, Olafsson JH, Steinsson JT, Kerrouche N, Sidou F.
J Eur Acad Dermatol Venereol. 2010 Aug;24(8):910-5. doi: 10.1111/j.1468-3083.2009.03547.x. Epub 2009 Dec 17.

Background: Standard treatment for onychomycosis often results in less than half of subjects achieving diseasefree nails. Onychomycosis is even more challenging to treat as relapses and re-infections are common.
Objective: To determine if a prophylactic effect exists when a treatment with amorolfine nail lacquer (ANL), with half the frequency of the standard regimen, is instituted following successful treatment of dermatophytic toenail onychomycosis with matrix involvement.
Methods: Efficacy and safety of a group treated with ANL (once every 2 weeks) were compared with that of an untreated group in a 36-month (3 years), single-centre, randomized, open-label, comparison study. Subjects to be included in the study were required to be cured of confirmed onychomycosis with matrix involvement after an initial treatment with either ANL + oral terbinafine or oral terbinafine alone in a previous study. Prophylaxis of onychomycosis was assessed by global recurrence rate, confirmed onychomycosis, clinical recurrence and mycological recurrence.
Results: A total of 52 subjects were enrolled (26 in each group) in the study. Throughout the study, recurrences occurred more quickly in the untreated group compared with that in the ANL group. Statistically significant differences were observed at month 12 (ANL, 8.3%; untreated, 31.8%; P = 0.047). At endpoint, 70.8% of the subjects treated with ANL remained cured compared to 50% in the untreated group (P = 0.153). Recurrence was delayed by nearly 200 days for the ANL group compared with that of the untreated group. Amorolfine was safe and well tolerated during the study, with no treatment-related adverse events.
Conclusion:These results suggest that amorolfine nail lacquer may be effective and is safe for use as a prophylactic treatment for the recurrence of onychomycosis.
Received: 7 August 2009; Accepted: 19 November 2009.

The role of biofilms in onychomycosis.
Gupta AK, Daigle D, Carviel JL.
J Am Acad Dermatol. 2016 Jun;74(6):1241-6. doi: 10.1016/j.jaad.2016.01.008. Epub 2016 Mar 22.

Onychomycosis is a fungal infection of nails primarily caused by dermatophyte fungi. Fungi are traditionally understood as existing in the environment as planktonic organisms; however, recent advancements in microbiology suggest that fungi form biofilmsecomplex sessile microbial communities irreversibly attached to epithelial surfaces by means of an extracellular matrix. The extracellular matrix also acts as a protective barrier to the organisms within the biofilm. The biofilm is surprisingly resistant to injury and may act as a persistent source of infection possibly accounting for antifungal resistance in onychomycosis.

In vitro characterization of Trichophyton rubrum and T. mentagrophytes biofilms.
Costa-Orlandi CB, Sardi JC, Santos CT, Fusco-Almeida AM, Mendes-Giannini MJ.
Biofouling. 2014;30(6):719-27. doi: 10.1080/08927014.2014.919282. Epub 2014 May 23.

Dermatophytes are fungi responsible for a disease known as dermatophytosis. Biofilms are sessile microbial communities surrounded by extracellular polymeric substances (EPS) with increased resistance to antimicrobial agents and host defenses. This paper describes, for the first time, the characteristics of Trichophyton rubrum and T. mentagrophytes biofilms. Biofilm formation was analyzed by light microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) as well as by staining with crystal violet and safranin. Metabolic activity was determined using the XTT reduction assay. Both species were able to form mature biofilms in 72 h. T. rubrum biofilm produced more biomass and EPS and was denser than T. mentagrophytes biofilm. The SEM results demonstrated a coordinated network of hyphae in all directions, embedded within EPS in some areas. Research and characterization of biofilms formed by dermatophytes may contribute to the search of new drugs for the treatment of these mycoses and might inform future revisions with respect to the dose and duration of treatment of currently available antifungals.

Our current understanding of fungal biofilms.
Ramage G, Mowat E, Jones B, Williams C, Lopez-Ribot J.
Crit Rev Microbiol. 2009;35(4):340-55.

Fungal biofilms are an escalating clinical problem associated with significant rates of mortality. Candida albicans is the most notorious of all fungal biofilm formers. However, non-Candida species, yeasts such as Cryptococcus neoformans, and filamentous moulds such as Aspergillus fumigatus, have been shown to be implicated in biofilm-associated infections. Fungal biofilms have distinct developmental phases, including adhesion, colonisation, maturation and dispersal, which are governed by complex molecular events. Recalcitrance to antifungal therapy remains the greatest threat to patients with fungal biofilms. This review discusses our current understanding of the basic biology and clinical implications associated with fungal biofilms.

Fusarium spp. is able to grow and invade healthy human nails as a single source of nutrients.
Galletti J, Negri M, Grassi FL, Kioshima-Cotica ÉS, Svidzinski TI.
Eur J Clin Microbiol Infect Dis. 2015 Sep;34(9):1767-72.

Onychomycosis caused by Fusarium spp. is emerging, but some factors associated with its development remain unclear, such as whether this genus is keratinolytic. The main aim of the present study was to evaluate the ability of Fusarium to use the human nail as a single source of nutrients. We also performed an epidemiological study and antifungal susceptibility testing of Fusarium spp. that were isolated from patients with onychomycosis. The epidemiological study showed that Fusarium species accounted for 12.4 % of onychomycosis cases, and it was the most common among nondermatophyte molds. The most frequent species identified were F. oxysporum (36.5 %), F. solani (31.8 %), and F. subglutinans (8.3 %). Fluconazole was not active against Fusarium spp., and the response to terbinafine varied according to species. Fusarium was able to grow in vitro without the addition of nutrients and invade healthy nails. Thus, we found that Fusarium uses keratin as a single source of nutrients, and the model proposed herein may be useful for future studies on the pathogenesis of onychomycosis.