According to the results of the clinical trials [6, 9,10,11], laser therapy is effective for the treatment of onychomycosis, but the in vitro findings are inconsistent among studies [14,15,16,17]. Therefore, this study aimed to explore the experimental conditions of the 1064-nm Nd:YAG laser for the inhibition of T. rubrum growth in vitro. Our results suggest that when the irradiation area was less than 6 mm, the 1064-nm Nd:YAG laser at 408 or 600 J/cm2 can effectively suppress T. rubrum growth in vitro totally or partially.
T. rubrum has a suitable growth temperature of 25–28°C. This study found that at 408 J/cm2 with 200 spots over a small area, the growth of T. rubrum was inhibited for a week when the temperature of the colony after laser irradiation exceeded the optimal growth temperature of T. rubrum. These results may be significant in guiding clinical treatment. Indeed, for the treatment of onychomycosis, when the surface temperature of the infected nails approaches the temperature observed in the present study, or even reaching 40–50°C, as in some studies [19, 20], the growth of the nail fungi should be inhibited for a week. Therefore, the treatment interval should be shortened. Currently, most clinical protocols apply a weekly dose [10, 21, 22]. So according to the study, we speculate whether it is possible to change the clinical protocol and shorten the interval. For example, the laser treatment should be given twice a week and to increase the energy density as much as possible. Using the proper laser, this can be achieved in terms of operability and feasibility, and the only problem may be the higher number of visits to the hospital. Of course, further clinical trials are needed to confirm our assumption. At 300 J/cm2 on a small area, the immediate temperature was still within the range of the optimal temperature for fungal growth, so it had not an inhibitory effect. At 600 J/cm2, the surface temperature of the colony exceeds the growth temperature of the fungus. While increasing the irradiation area, irrespective of energy, no growth inhibition effect was observed. As for why laser irradiation had no inhibitory effect on the large-area colony, we thought that a single session of laser irradiation cannot make the temperature high enough to inhibit the fungus. Even if when the energy density was 600J/cm2, the immediate temperature of the colony only reached 31°C and it dropped to the suitable growth temperature after 4 minutes, so there was no inhibitory effect.
Vural et al.  used a 1064-nm Q-switched Nd:YAG laser at 4 and 8 J/cm2 and a 532-nm Q-switched Nd:YAG laser at 8 J/cm2 to irradiate T. rubrum, and then observed the growth area of the colonies on the first, third, and the sixth day after irradiation. They found that the growth of T. rubrum was significantly inhibited. Ghavam et al.  found in an in vitro study that the laser with a lower energy density, like the red-blue light laser, near-infrared semiconductor laser and laser-diode pumped green laser, did not inhibit the growth of fungi, while the higher power lasers, like the 532-nm Q-switched Nd:YAG laser at 8 J/cm2, the Q-switched Nd:AG 1064-nm laser at 4–8 J/cm2 and the flashlamp-pumped dye laser at 8–14 J/cm2, could inhibit or kill T. rubrum. On the other hand, negative results were obtained by Hees et al. , who irradiated clinical cultures of T. rubrum with several types of lasers at different energy (1064-nm Q-switched Nd:YAG laser at 4 and 8 J/cm2, 532-nm Q-switched Nd:YAG laser at 8 J/cm2 and 1064-nm long-pulsed Nd:YAG laser at 45 and 100 J/cm2). Kim et al.  irradiated five clinical strains of T. rubrum with a long-pulse 1064-nm Nd:YAG laser for 0.3 ms at energy of 5 J/cm2 on 6 mm, every 3–5 days. On the 29th day, there was no significant difference in the color and area of the colonies, and they concluded that the laser had no inhibitory effect on T. rubrum. The lack of agreement on the inhibitory effect of laser on fungi may be because the energy density is not large enough, or the number of pulses is not enough. Therefore, the total energy is not sufficient, so there is no significant impact on the fungus. When using high energy density, it is possible that the number of pulses is sufficient. In the present study, we used a high energy density Nd:YAG laser and a killing effect was achieved at 408 and 600 J/cm2, but not at 300 J/cm2. Similar clinical effect was observed using higher energy densities [6, 11,12,13].
The inhibitory effects of laser irradiation on fungi are thought to be related to nonspecific thermal damage and the sensitivity of fungus to produced pigments to light sources. T. rubrum contains xanthomegnin, a dominant diffusing red pigment that absorbs laser irradiations at wavelengths of 532 and 598 nm . Therefore, laser irradiation at 532 or 598 nm has inhibitory effects on T. rubrum . Although the wavelength of the 1064-nm Nd:YAG laser exceeds the absorption spectrum of xanthomycin, similar inhibitory effects were observed on colonies treated at this wavelength. This may be due to absorption by another chromophore at 1064 nm, e.g., melanin, which is found in the cell wall of conidia [14, 23, 24]. Although T. rubrum produces melanin, this study found no obvious pigment production at the early stage, especially when the colonies were 6- or even 13-mm in diameter. With the growth of the colonies, the pigment gradually appeared. These findings indicated that the pigment may not play a critical role in response to laser irradiation, at least in vitro, and that the thermal coagulation effect is more important. It can be inferred from this study that the inhibitory effects of laser irradiation on fungi are possibly more associated with energy and exposure time: the higher the energy or the number of light spots administered, the higher the temperature of the fungi. When the temperature exceeds the fungus' tolerance threshold, its growth is inhibited, or death occurs. Therefore, when no pigment is produced, laser irradiation can also exert antifungal effects on T. rubrum. The possible mechanism is that high power lasers generate heat and increase the kinetic energy of the target cells, inducing death through evaporation, coagulation, or necrosis [7, 25].
The present study showed that the 1064-nm Nd:YAG laser had an inhibitory effect on T. rubrum. The present study and previous negative studies [16, 17] suggest some points for the optimization of the irradiation conditions. First, the in vitro environment does not accurately represent the in vivo conditions. The culture medium is extremely rich in nutrients, and the numbers of fungi are usually large, which are different from the in vivo environment. Although the laser inhibits or kills some fungi at the moment of treatment, the effect doses do not linger, and fungus growth can be restored if there are some fungi left. This is different from the effects of drugs. Second, the colony area at the beginning of laser irradiation may be too large, and a single session of laser irradiation cannot make the surface temperature high enough to inhibit the fungus. Third, the number of fungi on the plate is much larger than that found in infected nails. It was not possible to perform laser irradiation in vitro that completely mimics the clinical treatment parameters. Fourth, the infected nails generally have a color, and the therapeutic target of the laser is mostly pigments. The fungus cultured in the medium had not produced significant pigments at the early stage, which could affect laser efficacy. Fifth, the technique or skill of laser irradiation is also a factor. The fungi grow in a radial manner. Therefore, the area to be irradiated needs to be slightly larger than the actual area of the colony. If only the central part is irradiated, the peripheral hyphae are not inhibited.
Taken together, the 1064-nm Nd:YAG laser has an inhibitory effect on T. rubrum, but the experimental conditions need to be explored, and they cannot completely be in accordance with the clinically recommended parameters. The factors to be considered include the area of the colony at the beginning of laser irradiation vs. the area that needs to be irradiated (generally, it should be slightly larger than the area of the colony), the laser energy, the number of light spots, the numbers of laser irradiation sessions, and the duration of each irradiation. It was only possible to inhibit the growth of fungi when the cumulative temperature reached a certain amount per unit time and unit area. This is supported by Liu et al. , who showed that the 1064-nm Nd:YAG laser is effective against T. rubrum and that energy density and treatment times are the main factors involved in the effectiveness. Unfortunately, the exact energy output during laser irradiation could not be measured in the present study. Additional studies are still necessary to determine the best conditions for the laser treatment of T. rubrum.