| Objective:The purpose of the basic study was to observe the effects of long-pulse1064nmNd:YAG laser irradiation on the growth and ultrastructure changes of5mainpathogenic fungi of onychomycosis including Trichophyton rubrum, Trichophytonmentagrophytes, Candida albicans, Candida parapsilosis, Aspergillus fumigatus.And to compare drug sensitivity of some pathogenic fungi (e.g. C. albicans)beforeand after laser irradiation.The purpose of the clinical part was to have an initial evaluation on the clinicalefficacy, mycological clearance and safety of long pulse1064nm Nd:YAG lasertreatment for onychomycosis.Methods:PartⅠ:To observe the effects of long-pulse1064nm Nd:YAG laser irradiation on thegrowth of different pathogenic fungi, suspension of T. rubrum、T. mentagrophytes、C. albicans、C. parapsilosis were spotted on ager media. After incubation for severaldays, the colonies were exposed to various energies of1064nm Nd:YAG laser. Oneuntreated colony in each plate was left as the control. For T.r and T.m, diameters ofcolonies were measured on the first, third, and sixth days after laser irradiation. For C.a and C. p, the subculture’s survival rates of different energies of laser irradiationwere assessed respectively.As for A. fumigatus, having been incubated on the whole plate for one day,received various energies of Nd:YAG laser irradiation. One day after irradiation, thelaser treated spots exhibited circular growth-free zones, which differed in sizedepending on the energy delivered. we compared the different diameters of circulargrowth-free zones after different energies of laser treatment.At the same time, to observe the ultrastructure changes of the five pathogenicfungi, we scanned strains after various energies of1064nm Nd:YAG laser irradiationby scanning electron microscope (SEM) and transmission electron microscope (TEM).To explore if the laser irradiation had some effect on the drug sensitivity ofpathogenic fungi. we chose C.a strain Y01-09as an example, and detected the MICsof ltraconazole, fluconazole, and caspofungin of this strain before and after laserirradiation by referencing CLSI-M27A3.Part Ⅱ:There are37affected nails from14patients with onychomycosis were enrolledin our clinical study. All the14patients were mycologically proven fungal infectionbefore receiving long pulse1064nm Nd:YAG laser therapy. According to the ScoringClinical Index of Onychomycosis, we divided the37nails into different severities,and given laser therapy of six to eight sessions with one week interval. The lasertherapy used the following parameters:35to40J/cm2fluence,35ms pulse duration,4mm spot size,1Hz frequency. In order to fully cover the nail plate, we given fourpasses across each nail plate in one session. Follow up was performed at2,6, and10months after first laser therapy and the clinical efficacy was evaluated at the sametime. In order to assess mycological clearance, mycological tests includingmicroscopic examination and culture were performed before irradiation and twomonths after first laser therapy. Side effects(e.g. local pain, et al.) were observedthroughout therapeutical period and the follow-up.Results:PartⅠ:For T.r or T.m, compared with the control, the colonies’ diameters weresignificantly smaller(P＜0.05) when the laser energy reached3200J/cm2or more,respectively. However within those groups, when laser energy reached about6400J/cm2,12800J/cm2,25600J/cm2, the diameters of the colonies had no significantdifference(P＞0.05).For C. a and C. p, when the laser energy being more than3200J/cm2, all thesubculture’s survival rates were lower than their each control (P＜0.05).For A.fumigatus, when the laser energy being more than3200J/cm2,the growthwas inhibited significantly. And the higher the laser energy was, the bigger the diameter of circular growth-free zones would be.(P＜0.05for all)Compared with the control, these five pathogenic fungi presented different degreeof ultrastructure damages when laser energy reached at3200J/cm2,6400J/cm2,12800J/cm2.In drug sensitivity tests of C. albicans Y01-09to itraconazole, fluconazole, andcaspofungin before and after laser irradiation, C. albicans strain Y01-09of controlgroup,800J/cm2group and1600J/cm2group showed nearly the same drug sensitivityprofiles.However C. albicans strain Y01-09of3200J/cm2group did not grow inevery well with or not with antifungal agents.PartⅡ:Thirty-seven nails of fourteen patients have been treated using the Nd:YAG1064nm laser for six to eight sessions. The effective rates at2,6,10months after firstlaser therapy were40.54%、48.65%、56.76%. And the mycological clearance was78.57%at two months after first laser therapy. During the whole treatment, most ofthe patients felt warm in the local areas, some of them complained temporary mildsearing pain which could be well tolerated. No adverse reaction was found duringfollow-up period.Conclusion:In vitro, when the energy of long pulse1064nm Nd:YAG laser reaching a certainamount, the pathogenic fungi of onychomycosis could be inhibited significantly or bekilled, and the ultrastructures of them also showed different degree of damagesaccordingly.The drug sensitivity of C. albicans strainY01-09to itraconazole, fluconazole, andcaspofungin did not show difference within the unexposured group and several lowerenergy exposured groups. Due to the lowest growth activity of fungi, microdilutionantifungal susceptibility testing did not work in higher energy exposured groups,within which the strainY01-09could not even grow in wells with nomal liquid media.Long pulse1064nm Nd:YAG laser was effective for onychomycosis of differentseverities. It’s a simple, effective, and safe therapeutic method without noticeable sideeffects. Such therapeutic method offers a new alternative treatment for onychomycosis cases, and is specially beneficial in elderly patients, patients with lowimmunity, liver and renal dysfunction. |
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