TREATMENT OF FUNGAL INFECTIONS

Abstract

The present disclosure provides compositions, medicaments and methods for treating or preventing disorders, conditions and/or diseases affecting keratinised structures or tissues such as, for example, nails.

Claims

1. A method of treating or preventing a fungal infection, said method comprising administering a subject suffering from, or predisposed to a fungal infection, an immunomodulatory amount or dose of microwave energy.

2. The method of claim 1, wherein the fungal infection is a fungal infection of a keratinised tissue or keratinised structure.

3. The method of claim 1, wherein the fungal infection is an onychomycosis condition of a nail.

4. The method of claim 1, wherein the fungal infection is caused or contributed to by any one or more of the below listed fungal pathogens: (i) a dermatophyte; (ii) a Fusarium; (iii) a Microsporum sp.; (iv) a Michrodochium sp.; (v) a Epidermophyton sp.; (vi) a Trichophyton sp.; (vii) Trichophyton rubrum; (viii) Trichophyton mentagrophytes; and (ix) Epidermophyton floccosum.

5-7. (canceled)

8. The method of claim 1, wherein the subject is immunocompromised.

9. The method of claim 1, wherein the immunomodulatory dose does not kill or inactivate the fungal pathogen.

10-11. (canceled)

12. The method of claim 1, wherein the microwave energy is administered to modulate the activity, expression and/or function of the host innate and adaptive immune response and/or to modulate the expression, function and/or activity of those host immune factors, cytokines, chemokines, inflammatory factors, anti-inflammatory factors and/or apoptotic factors which are modulated by the fungal pathogen.

13-15. (canceled)

16. A method of downregulating one or more of: (i) PI3K; (ii) p-AKT; (iii) IL-10; (iv) EGR1; (v) CD79B; (vi) MYC; and/or (vii) SOCS3; in a subject, said method comprising administering the subject microwave energy.

17. A method of upregulating one or more of: (i) IL-1β; (ii) IL-6; (iii) IL-2; (iv) IL-12; (v) (TNF)-α; (vi) IFN-γ; (vii) MHC class 2 APC; (viii) CD80; (ix) CD74; (x) CD4; (xi) T cell receptor; (xii) NFkB; (xiii) IL-18; (xiv) MAPK1; (xv) HSP; and (xvi) MHC class 1. in a subject, said method comprising administering the subject microwave energy.

18-22. (canceled)

23. The method of claim 1, wherein the microwave energy has a frequency of between about 500 MHz and about 200 GHz, or about 900 MHz and about 100 GHz, or about 900 MHz and about 100 GHz, or a frequency of 8 GHz.

24-26. (canceled)

27. The method of claim 1, wherein the microwave energy is administered as a single continuous dose or as a pulse dose comprising multiple, spaced apart individual doses.

28. The method of claim 1, wherein the microwave energy is administered at a power of 1 W, 2 W, 3 W, 4 W, about 5 W, 6 W, 7 W, 8 W, 9 W, 10 W, 11 W or 12 W.

29. The method of claim 1, wherein the microwave energy is administered for 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds or 10 seconds.

30. The method of claim 27, wherein the multiple spaced apart doses are separated by a rest period of 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds or 10 seconds.

31. (canceled)

32. The method of claim 27, wherein the microwave dose is repeated.

33. (canceled)

34. The method of claim 32, wherein each repeated dose is separated from another by a dose rest period.

35. (canceled)

36. The method claim 1, wherein the microwave energy is administered as a dose regime comprising three individual doses of 9W with five seconds rest between each individual dose.

37. The method of claim 36, wherein the dose regime is repeated or repeated five times over a period of 10 weeks.

38. (canceled)

39. The method of claim 1, wherein the microwave energy is administered as a dose regime comprising an initial dose of microwave energy designed to elevate the temperature of the area, tissue or structure being treated and a second dose designed to maintain a temperature in the area, tissue or structure being treated.

40. The method of claim 39, wherein the initial dose may comprise microwave energy at a power of 10 W, 15 W, 20 W or 25 W.

41. The method of claim 39, wherein the second dose may comprise microwave energy at a power of 1 W, 2 W, 3 W, 4 W, 5 W, 6 W, 7 W, 8 W, 9 W or 10 W.

42. The method of claim 39, wherein the first dose is administered for anywhere between 5 and 30 seconds.

43. The method of claim 39, wherein the second dose is administered for anywhere between 5 and 30 seconds.

44. The method of claim 39, wherein the first dose raises the temperature of the area, tissue or structure being treated to between about 30° C. and 60° C.

45. The method of claim 39, wherein the second dose raises the temperature of the area, tissue or structure being treated to between 20° C. and 60° C.

46. The method of claim 1, wherein the microwave energy is administered directly to the site of infection.

47. The method of claim 1, wherein the microwave energy is administered to the same or a different site.

48. The method of claim 1, wherein the microwave energy is administered to (i) the site of the fungal infection; (ii) a site proximal or adjacent to a fungal infection; or (iii) a site distal to the site of a fungal infection.

49. The method of claim 45, wherein the site to be treated is first hydrated or moistened.

50. The method of claim 49, wherein hydrating or moistening the site or sites to be treated comprises immersing the site or sites to be treated in a water bath to hydrate or moisten the site or sites.

51. The method of claim 49, wherein the site(s) to be treated is/are a fingernail(s) or toenail(s).

52. The method of claim 49, wherein the step of hydrating or moistening the site to be treated, includes or comprises the use of a hydration and/or lossy ionic fluid.

53. The method of claim 52, wherein the hydration and/or lossy ionic fluid buffers the energy closer to the surface of the site to be treated or within certain predetermined or defined regions of the site and/or its surrounding tissues and structures.

54. The method of claim 52, wherein the hydration and/or lossy ionic fluid comprises sodium chloride (NaCl).

55. The method of claim 48, wherein the site to be treated is pre-treated with Urea.

56. The method of claim 55, wherein the urea is applied to the site to be treated at a concentration of 10% to 50%.

57. The method of claim 55, wherein a urea pre-treatment is administered to the site to be treated before the site to be treated is hydrated or moistened.

58-67. (canceled)

68. The method of claim 1, wherein the microwave energy is administered to a or the keratinised structure using an apparatus comprising a microwave energy source and a microwave applicator formed, adapted and/or configured to deliver or administer microwave energy to the subject.

69. The method of claim 68, wherein the microwave energy, the applicator or the means of administering or delivering the microwave energy electrically matches the range of epsilon relative values of the keratinised structure to be treated.

70. The method of claim 68, wherein the applicator may be lubricated, coated or used with a buffer medium to remove airgap discontinuity between the applicator contacting and the keratinised structure to ensure good delivery of microwave energy.

71. The method of claim 70, wherein the buffer medium comprises propylene glycol, glycerine.

72. The method of claim 68, wherein the microwave energy source produces microwave energy at a single frequency and/or microwave energy across a range of frequencies.

73-85. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0167] FIG. 1 (upper and lower panels). Nail comprises the nail plate (corpus unguis) 1, the Lateral horns (Lunula) 2, the nail root (germinal matrix), (radix unguis) 3, the lateral nail fold (Paronychium), 4, the quick (Hyponychium) 5, the nail bed (sterile matrix) 6, the cuticle (Eponychium) 7, the nail cleft (sinus unguis) 8 the periosteum 9 and the ventral floor 10.

[0168] FIG. 2 depicts a microwave power generator system for medical applications. The apparatus comprising: —a microwave source for providing microwave energy 11, connectable to a system controller 12 for controlling at least one property of the microwave radiation provided by the microwave source; and a monitoring system 13 for monitoring the delivery of energy and an interconnecting cable 14 and an applicator hand piece 15 and a removable applicator means 16, for example an applicator device, for delivering microwave energy, wherein: —the applicator is configured to deliver precise amounts of microwave energy provided by the source at a single frequency or across a range of frequencies.

[0169] FIG. 3 depicts a schematic representation of a cellular signalling system. In this illustration the main immune interactions between the host and Trichophyton Rubrum 17 are summarised.

[0170] FIG. 4 shows the components of an apparatus according to the present disclosure, the components shown separately for ease of reference. The apparatus comprises a generator system 24 with a locking microwave connection 25 to a flexible microwave cable 26 connected to a hand piece 27 (which may have the same type of locking connection) which accepts an applicator component 28.

[0171] FIG. 5 shows an embodiment of a microwave treatment for Onychomycosis. In this embodiment a microwave applicator 29 is introduced above the nail plate (corpus unguis) and microwave energy is applied to the nail structure producing targeted tissue irradiation 30 to a depth 31 determined by the frequency of application in conjunction with the properties of the nail/tissue structure. The microwave frequency chosen will be sufficient that the penetration of energy will be limited to the to prevent damage to the underlying tissues such as the nail bed (sterile matrix) 6, and the periosteum 9.

[0172] FIG. 6 depicts an Onychomycosis infection which covers a significant proportion of the nail plate 32, various treatment application regimens may be used to cover the required area of the infected nail. This may be done as a piecewise application 33 where overlapping treatment areas may be selected. This may be further enhanced by using a thermochromic barrier or coating 34 that may be affixed to the nail to indicate regions already treated. Similarly, the treatment may comprise a continuous sweeping motion from left to right and reversed from right to left 35 or from proximal to distal and reversed from distal to proximal 36.

[0173] FIG. 7 depicts an Onychomycosis infection which covers a significant proportion of the nail plate, various treatment application regimens may be used to cover the required area of the infected nail. For example, the piecewise application 37 may occur from proximal to distal in a series of linear steps 38 or in a series of linear sweeps 39 each in the same left to right or right to left direction 40 or as a series of linear sweeps 41 from proximal to distal 42 or from distal to proximal.

[0174] FIG. 8 depicts a number of simulated results (COMSOL finite element analysis) are presented for various nail properties.

[0175] FIG. 9 depicts a number of simulated results (COMSOL finite element analysis) are presented for various nail properties.

[0176] FIG. 10 depicts Trichophyton rubrum spores were cultured in agar petri dishes.

[0177] FIG. 11 depicts Trichophyton rubrum spores were cultured in agar petri dishes after microwave (MW) treatment.

[0178] FIG. 12 is a graph illustrating the mean area (mm.sup.2) of clearance in culture following microwave treatment for each protocols.

[0179] FIG. 13 depicts an Onychomycosis case prior to treatment with microwave energy.

[0180] FIG. 14 depicts the Onychomycosis case of FIG. 13 1 month after treatment with microwave energy.

[0181] FIG. 15 depicts the Onychomycosis case of FIG. 13 6 month after treatment with microwave energy.

[0182] FIG. 16 depicts the Onychomycosis case of FIG. 13 10 month after treatment with microwave energy.

[0183] FIG. 17 depicts the Onychomycosis case of FIG. 13 10 month after treatment with microwave energy, specifically distal outgrowth of the nail.

DETAILED DESCRIPTION OF THE INVENTION

[0184] The anatomy of the nail is illustrated in FIG. 1 (upper and lower panels). Nail comprises the nail plate (corpus unguis) 1, the Lateral horns (Lunula) 2, the nail root (germinal matrix), (radix unguis) 3, the lateral nail fold (Paronychium), 4, the quick (Hyponychium) 5, the nail bed (sterile matrix) 6, the cuticle (Eponychium) 7, the nail cleft (sinus unguis) 8 the periosteum 9 and the ventral floor 10.

[0185] An embodiment of a microwave power generator system for medical applications is illustrated in FIG. 2 The apparatus comprising: —a microwave source for providing microwave energy 11, connectable to a system controller 12 for controlling at least one property of the microwave radiation provided by the microwave source; and a monitoring system 13 for monitoring the delivery of energy and an interconnecting cable 14 and an applicator hand piece 15 and a removable applicator means 16, for example an applicator device, for delivering microwave energy, wherein: —the applicator is configured to deliver precise amounts of microwave energy provided by the source at a single frequency or across a range of frequencies.

[0186] FIG. 3 provides a schematic representation of a cellular signalling system. In this illustration the main immune interactions between the host and Trichophyton rubrum 17 are summarised.

[0187] The intracellular enzyme Phosphoinositide 3-kinase PI3-K has been linked to a range of intracellular functions for example, cell growth, differentiation, proliferation, motility, survival and trafficking. PI3-K typically activates protein kinase B (PKB, aka Akt) in the PI3-K/AKT/mTOR pathway. PI3K/Akt is also known to play an important role in mediating survival of monocytes/macrophages in response to signalling factors and cytokines and in blocking apoptosis by toxic invading organisms. PI3K increases anti-inflammatory cytokine production and has also been known to offer a mediated protection against epithelial damage during infection by some fungal species for example Candida albicans [′] and has been implicated in the anti-apoptotic property of a variety of pathogens. For example, sustained PI3K/Akt signalling [.sup.ii] in cells infected with human pathogen Aspergillus fumigatus can exert a cytoprotective effect enabling infected macrophages to resist apoptosis. Although the evidence for this occurring with Trichophyton rubrum 19 is limited, modulation of this pathway is anticipated based upon its involvement with other fungal pathogens.

[0188] Referring to the lower panel in FIG. 3, microwave energy 20 applied to tissue infected with a fungal pathogen, may cause a down regulation of (PI3-K) 21 which regulates cellular signalling. This may be achieved by any of the microwave energy doses described herein, including the pulsed or continuous microwave energy-based methods. An inhibitor of PI3K will decrease anti-inflammatory cytokine production, and this downregulation has been achieved with the use of microwave energy downregulating the PI3K/PIK3R2 signalling pathway.

[0189] It has also been observed [Error! Bookmark not defined.] in patients with chronic widespread dermatophytosis (CWD) due to Trichophyton rubrum that their macrophages secreted lower amounts of pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α 18, however these patients also expressed increased levels of anti-inflammatory cytokine IL-10.

[0190] The signalling and/or expression of these cytokines can be modulated (for example, reversed) by the application of microwave energy, this upregulates at least interleukin (IL)-1β, IL-6, ad tumor necrosis factor (TNF)-α, and down-regulates expression of the anti-inflammatory cytokine IL-10.

[0191] Trichophyton rubrum has also been seen [iii] to act as a downregulator of class II major histocompatibility complex (MHC) antigens 18 and in the expression of co-stimulatory molecules. It has now been shown that MHC antigen expression and Interleukin (IL)-18 expression (an immune-enhancing cytokine, which induces Interferon (IFN)-γ which in turn activates macrophages) can be upregulated by administration of microwave energy as disclosed herein. It should be noted that upregulation of IFN-gamma is linked to the up-regulation of MHC class I expression and has a positive effect on antigen processing and presentation. Microwave energy (via modulation of IFN-gamma) has also been shown to have an effect on MHC class II expression induces.

[0192] IFN-γ has been shown [iv] to impair Trichophyton rubrum proliferation through the production of reactive oxygen species (ROS). Moreover, IL-12 and IFN-γ are influential in controlling the infection and both these factors and have now been shown to be upregulated by microwave energy for therapeutic benefit.

[0193] In observed [v] interactions between Trichophyton rubrum conidia with macrophages, the expression of (TNF)-α and IL-10 were modulated (but not IL-12 and nitric oxide). Infected macrophages downregulated the expression of co-stimulatory molecules (CD80 and CD54). Microwave energy application can increase expression of heat shock proteins (HSPs) enhancing T-cell activation resulting in subsequent upregulation of CD80, CD86, CD74 and CD4. Dermatophytes including Trichophyton rubrum can also intrinsically express HSP70 and HSP90 in response to physiological stresses [vi, vii] and HSP90 plays a crucial role in the development of antifungal resistance of Candida albicans to both of the main antifungals, the echinocandins and the azoles. Since it has been shown that microwave energy induces an HSP response in tissue it may also be used to induce a similar response in the dermatophyte with this being used to exhaust the protective influence of the dermatophyte's expression of HSP rendering the pathogen susceptible to the host's natural immune system defences. Microwave energy applied in brief pulses or for short intervals represents a unique and unnatural challenge to any organism and may be one that its evolved protection mechanisms are unequipped to combat. Accordingly, a method provided by this disclosure may exploit microwave energy as a way to upregulate expression of HSP70 and HSP90 in certain fungal pathogens. A method of this type may comprise administering microwave energy to any of the subjects described herein at a dose described herein.

[0194] Trichophyton rubrum has also been determined [viii] to increase the expression of TLR2, TLR4 and TLR6 and to induce HBD-1 and HBD-2 production. The innate immune function of keratinocytes as the initial stage of localised skin immunity is effectively influenced and adapted by Trichophyton rubrum ensuring the pathogen can become established and the infection can persist. Microwave energy may be used to modulate the function of keratinocytes and to reverse or counter the effects the dermatophyte has on the innate immune system.

[0195] The common antifungal drug terbinafine has been reported to inhibit the expression of the ERG1 and ERG11 genes [ix]. The ERG1 gene can be downregulated by microwave energy 23.

[0196] Increased CD79b(+) cells have been observed in feline models [x] with significant expression of class II antigens of the major histocompatibility complex associated with epithelium involvement near to fungal elements. Significantly, more CD79b(+) cells were found to have infiltrated the epithelium. Elevated levels of CD79B have also been reversed by application of microwave irradiation 23.

[0197] MYC [xi] is known to suppress immune surveillance, MYC [xii] also plays a primary role in the regulation of aerobic glycolysis. MYC directly activates the transcription of the majority of glycolytic genes. The glycolysis pathway is crucial for carbon assimilation and is upregulated during infections with pathogenic fungi such as C. albicans [xiii], Trichophyton rubrum [xiv] and Paracoccidioides brasiliensis [xv] It is known [xvi] that macrophages react to increased glycolysis by upregulating antimicrobial inflammation and by killing pathogens. In the case of Candida-macrophage interactions a combined metabolic change occurs, with concurrent upregulation of glycolysis in both host and pathogen establishing competition for glucose. As Candida can survive on multiple carbon sources it has an advantage over infected macrophages which are metabolically constrained to glycolysis and depend upon a glucose source for survival. Candida benefits from this constraint by exhausting the supply of available glucose thereby starving the macrophage of energy and establishing and advantage. Microwave energy 23 can be used to downregulate MYC 23 which may be used to reverse the glycolysis process local to dermatophyte infection.

[0198] It has been shown [xvii] that the suppression of SOCS3 in dendritic cells can enhance the activation and specialisation of dendritic cells. Suppression of SOCS3 can activate phagocytosis and killing of the fungal pathogen by the dendritic cells. Suppression of SOCS3 can also increase promote IL-6-induced tyrosine phosphorylation of STAT3 in dendritic cells and improved capability of DCs to prime naïve T cells and initiate Th17 proliferation which protects against intracellular fungal pathogens. Microwave energy has now been demonstrated to supress SOCS3 by a factor of almost 6× in tissue with elevated expression of this gene 23.

[0199] Another mechanism that may be exploited is the Ca2+ calcium signalling pathway [xviii] which can theoretically be modulated using pulsed application of microwave energy.

[0200] FIG. 4 shows the components of an apparatus according to the present disclosure, the components shown separately for ease of reference. The apparatus comprises a generator system 24 with a locking microwave connection 25 to a flexible microwave cable 26 connected to a hand piece 27 (which may have the same type of locking connection) which accepts an applicator component 28. The applicator component is designed to match to the combined tissue and material dielectric properties of the nail, pathogen infection and the nail bed 6. The cable 26 may include both microwave and signal data cables and may be reversible to enable connection to either port.

[0201] FIG. 5 shows an embodiment of a microwave treatment for Onychomycosis. In this embodiment a microwave applicator 29 is introduced above the nail plate (corpus unguis) and microwave energy is applied to the nail structure producing targeted tissue irradiation 30 to a depth 31 determined by the frequency of application in conjunction with the properties of the nail/tissue structure. The microwave frequency chosen will be sufficient that the penetration of energy will be limited to the to prevent damage to the underlying tissues such as the nail bed (sterile matrix) 6, and the periosteum 9.

[0202] In reference to FIGS. 6 and 7, where the Onychomycosis infection covers a significant proportion of the nail plate 32, various treatment application regimens may be used to cover the required area of the infected nail. This may be done as a piecewise application 33 where overlapping treatment areas may be selected. This may be further enhanced by using a thermochromic barrier or coating 34 that may be affixed to the nail to indicate regions already treated. Similarly, the treatment may comprise a continuous sweeping motion from left to right and reversed from right to left 35 or from proximal to distal and reversed from distal to proximal 36. Alternatively, the piecewise application 37 may occur from proximal to distal in a series of linear steps 38 or in a series of linear sweeps 39 each in the same left to right or right to left direction 40 or as a series of linear sweeps 41 from proximal to distal 42 or from distal to proximal. This application method may be further enhanced by utilising materials or compounds applied to the nail with lubricants embedded or added to reduce friction and facilitate this sweeping motion. It should be noted that any sweeping motion of an applicator across the surface of a nail to be treated, may be facilitated by the use of a lubricant. For example a lubricated film, barrier, fluid or compound may be coated, attached and/or deposited onto the surface to be treated (for example the surface of a nail, toenail or fingernail) to enhance travel of an applicator across the that surface.

[0203] In FIG. 8, a number of simulated results (COMSOL finite element analysis) are presented for various nail properties. Firstly, a nail with an underlying air-gap 43 is considered. This often occurs in an Onychomycosis infection where the distal nail detaches from the nail bed due to the underlying fungal invasion. In this scenario it can be seen in the case of a dry nail 44 that applied microwave energy can permeate more readily into the underlying tissues of the nail bed. Where the nail has been moistened 45 it can be seen via simulation that the energy can be further constrained within the nail region. This is further developed for a hydrated 46 nail where the energy is further constrained to this region with less energy permeating into the underlying tissue. The wet nail may be achieved by use of a water bath to moisten the nails prior to treatment. In the case of hydrated nail this may be achieved by application of urea (10-50%) prior to the treatment (one or more days before) to increase the retained moisture within the nail. In each case the dielectric constant and electrical loss tangent increase with increased moisture content. This increases the heating and focuses the energy within a smaller zone within and proximate to the nail. This enhances the energy density and reduces heating of the underlying tissue which makes for a more easily tolerated treatment without the requirement for local anaesthetic.

[0204] This approach can also be enhanced by the application of Sodium chloride (NaCl) 47 to each nail example. In the case of the dry nail 48, saline may be injected or deposited under the nail to take up the air gap. In the case of the wet nail 49 saline may be injected or deposited under and around the nail to take up the air gap. In the case of the hydrated nail, fluid with a high salinity can be added under the nail to constrain the energy significantly within the nail structure. These methods may have advantages where the Onychomycosis infection results in dry or brittle nails. In those cases, the energy may pass through the nail and fungal regions into the nail bed causing excessive heating of the derma tissues rather than the desired fungal infected regions. In this regard, hydration and/or lossy ionic fluids or materials may be employed to buffer the energy closer to the surface or within desired regions of the nail structure. In addition, low microwave loss fluids such as distilled or de-ionized water (including chilled fluids) can conversely be used to limit the heating effect of the microwave radiation in regions intended to be protected or preserved.

[0205] In order to examine the direct effect of microwave energy on Trichophyton rubrum fungus, Trichophyton rubrum spores were cultured in agar petri dishes as shown in FIG. 10. Microwave energy at 8 GHz was applied to the culture from below using the SWIFT microwave system. A number of energy application protocols were trialled and the culture was monitored for 10 days to document reaction to this energy. There were 4 distinct areas/segments of energy application each receiving energy with different protocol parameters. From the underside the 1.sup.st region 52 corresponded to H-50 on the top-side 60, the 2.sup.nd underside region 53 corresponded to M-44 on the top-side 59, the 3.sup.rd underside region 53 corresponded to L-41 on the top-side 58 and the 4.sup.th underside region 55 corresponded to P-50 on the top-side 57. There was also a control region C 56 which did not receive any energy.

[0206] In the photograph in FIG. 11 taken 5 days pending application of the energy a distinctive sunken area within the culture can be seen where the Trichophyton rubrum fungus has been disrupted from normal growth compared to the control region C 56.

[0207] The area of clearance was recorded each day and was found to peak approximately 6 to 7 days after the treatment before regressing due to fungal ingrowth from the treatment margin. It should be noted that no new growth occurred from within the treated zone so it was determined that untreated fungus at the margin regained this area as opposed to a recovery occurring inside the treated zone. This data is presented in FIG. 12 which illustrates the mean area (mm.sup.2) of clearance in culture following microwave treatment for each of the protocols. This data is further summarised in a table below.

TABLE-US-00005 Ref T1 T2 number Regimen Ramp (° C.) Hold (° C.) Segment 62 H-50 15 W 20 s 55 5 W 30 s 49-50 1 (circle) 63 M-44 15 W 13 s 46-47 5 W 20 s 44 2 (square) 64 L-41 20 W 10 s 41 5 W 20 s 41-42 3 (cross) 61 P-50 (20 W 42 (20 W 48-50 4 (triangle) 3 s) × 3 5 s) × 3 (pulsed) Control n/a n/a n/a n/a C Note: H-50 was 15 Watt/20 sec ramp 5 Watt/30 sec hold 62 (circles) applied to segment 1. M-44 was 15 Watt/13 sec ramp 5 Watt/20 sec hold 63 (squares) applied to segment 2. L-41 which was 20 Watt/10 sec ramp 5 Watt/20 sec hold 64 (crosses) applied to segment 3. P-50 which was 20 Watt/3 sec pulsed 3× times with a 20 Watt/5 sec hold pulsed 3× times 61 (triangles) applied to segment 4.

[0208] Temperatures were balanced to establish if there was a difference between pulsed or continuous energy application. It was found that for the same temperature during the hold phase that intermittently pulsed (seconds) microwave energy had a significant effect on the Trichophyton rubrum colony morphology resulting in the highest amount of clearance area. The energy levels required to achieve this also included the heating of the agar medium which necessitated higher power levels that could not be expected for treatment in human tissue due to the volume of material and distance travelled by the energy in the experimental media. The temperature levels however were translatable for human use.

[0209] As illustrated in FIG. 13 this treatment was applied to an Onychomycosis case by an independent clinician who secured the patients' approval to treat only with microwave energy. The baseline condition state was recorded in FIG. 13, In this case a “nail spike” of Onychomycosis infection was presented. The initial distance recorded between the infection and the proximal nail fold was 1.2 mm. In this state left to progress it could reasonably be assumed that without treatment the infection would eventually engulf the entire nail area including the proximal nail fold resulting in the destruction of the nail.

[0210] A distinctive tri-globular area 65 was observed as a focal point to monitor the progression of the infection. Pending treatment and upon follow-up at +1 month as illustrated in FIG. 14, the tri-globular area 66 was observed to have advanced towards the distal end of the nail with the area of separation between the advancing front of the Onychomycosis invasion and the proximal nail fold increasing to 1.4 mm.

[0211] Further treatment was provided and pending a long treatment hiatus the patient presented+6 months from the initial baseline with an improved state of the Onychomycosis condition of the nail. This is illustrated in FIG. 15 where a 3 mm separation 67 between the proximal nail fold and the advancing front of the Onychomycosis infections was evident. Interestingly a darker lateral margin was observed to have appeared between the healthy nail bed and the region of Onychomycosis and this was not observable in the baseline photographs.

[0212] The Patient received further treatment and returned+10 months from baseline with further improvement evident. In this observation the separation 68 between the proximal nail fold and the advancing front of the Onychomycosis infection increased to 4.5 mm of clear new nail. This trend would suggest that the nail should continue to grow out naturally and thus eliminate the fungal infection. It was however interestingly noted that the lateral margin 70 had advanced in the medial direction 69 in addition to the distal outgrowth of the nail as highlighted in FIG. 17. It must be assumed that the host immune system had mounted a response to the fungus and thus had succeeded in eliminated an area of fungal invasion in addition to preventing the advancement of the disease towards the proximal nail fold. The dark (red) margins 70 are suggestive of an inflammatory or reactive region that according to the aforenoted theory could be suppressed by an active Trichophyton rubrum fungal infection. It may be assumed that the fungus has been weakened or deactivated by microwave treatment to such an extent that it cannot exert any signalling advantage or influence over the hosts innate immune system and has thus started to be successfully repelled. The progress duration being the natural growth-rate of the nail which varies with health, age, nutrition and other factors. The active (alive) state of the fungus can also be established by culturing samples to attempt to propagate the fungus in-vitro.

[0213] In summary this approach has detailed both in-vivo and in-vitro successful control of a fungal organism using a unique microwave energy treatment modality.

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