Combined laser and ozone therapy for onychomycosis in an in vitro and ex vivo model

Abstract

In order to develop a fast combined method for onychomycosis treatment using an in vitro and an ex vivo models, a combination of two dual-diode lasers at 405 nm and 639 nm wavelengths, in a continuous manner, together with different ozone concentrations (until 80 ppm), was used for performing the experiments on fungal strains growing on PDA agar medium or on pig's hooves samples. In the in vitro model experiments, with 30 min combined treatment, all species are inhibited at 40 ppm ozone concentration, except S. brevicaulis, which didn't show an inhibition in comparison with only ozone treatment. In the ex vivo model experiments, with the same duration and ozone concentration, A. chrysogenum and E. floccosum showed total inhibition; T. mentagrophytes and T. rubrum showed a 75% growth inhibition; M. canis showed a delay in sporulation; and S. brevicaulis and A. terreus did not show growth inhibition. This combined laser and ozone treatment may be developed as a fast therapy for human onychomycosis, as a potential alternative to the use of antifungal drugs with potential side effects and long duration treatments.

Fig 1. Equipment designed for the combined ozone and laser therapy of onychomycosis, showing the central irradiated chamber (left) where the bag receiving the ozone gas is placed (right).

Fig 2. Percentage of viability for each of the eight fungal species with laser treatments using only 405 nm, only 639 nm and both lasers combined at three different times (10 min, 20 min and 30 min).

Statistical significant results are shown in comparison with the absolute control and indicated with asterisk.

Fig 3. Percentage of viability for each of the eight fungal species with ozone treatments (20 ppm, 40 ppm, 60 ppm and 80 ppm) at three different times (10 min, 20 min and 30 min).

Statistical significant results are shown in comparison with the absolute control and indicated with asterisk.

Fig 4. Percentage of viability for each of the eight fungal species with ozone treatment 40 ppm, in combination with both laser treatments simultaneously at three different times (10 min, 20 min and 30 min).

Statistical significant results of the combined ozone and laser treatments are shown in comparison with only ozone, only laser treatments or absolute control at each time point, and indicated with asterisk. Asterisks without a comparison line indicate the same statistical significant results between all conditions at the same time.

Fig 5. Percentage of viability for each of the eight fungal species with ozone treatment 60 ppm in combination with both laser treatments simultaneously at three different times (10 min, 20 min and 30 min).

Statistical significant results of the combined ozone and laser treatments are shown in comparison with only ozone, only laser treatments or absolute control at each time point, and indicated with asterisk. Asterisks without a comparison line indicate the same statistical significant results between all conditions at the same time.

Fig 6. Porcine hoof samples (1 cm²) inoculated with 10⁵ CFUs of one of the fungal strains, after 2 weeks incubation period under water-saturated atmosphere.

The white mycelium can be observed on the hoof surface.

Fig 7. Growth of the eight fungal strains on porcine hooves samples placed on PDA medium, after ozone treatments at 40 ppm or 60 ppm combined with both laser wavelengths, during 30 min.