An ultrastructural study of Trichophyton rubrum induced onychomycosis

Abstract

Background: Trichophyton rubrum (T.rubrum) caused onychomycosis is the most common nail fungal disease. The common diagnostic methods are direct microscopic examination and fungal culture. In this study we used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to study the subungual ultrastructural changes in T. rubrum induced onychomycosis.

Methods: Six outpatients with onychomycosis were recruited and T.rubrum infection was confirmed by fungal culture. Six toenail samples were collected and prepared for SEM characterization. The cultured fugal colonies were prepared for SEM and TEM characterization.

Results: 1) SEM showed significant structural damages and the formation of a thin layer or a single layer of keratinocytes in all infected nail plates. Hyphae (piercing or penetrating keratinocytes layers), arthrospores and local bacterial aggregation were observed on the ventral surface of the nail plates. 2) SEM of the cultured fungal colony showed relatively straight, highly branched hyphae and microconidias; TEM showed branching hyphae that were composed of double-layer cell walls. Hyphae had nucleus, mitochondria, liposomes, lysosomes, scattered rough endoplasmic reticulum, myeloid bodies and aggregated ribosomes. There were high-density particles outside the hyphae.

Conclusion: SEM showed a large number of hyphae penetrated the keratinocytes layer, suggesting that T. rubrum can cause severe damage to the stratum corneum. TEM showed the ultrastructural features of T. rubrum-induced infection before treatment.

Fig. 1

a. Case 1 had thickened nail plate, gray-black, visible subungual debris, and absence of distal nail plate. b. Case 2 had mild thickened nail plate, subungual yellow debris, yellow and uneven nail plate surface

Fig. 2

a. Light microscopic examination. Case 2 showed visible transparent hyphae in subungual debris (arrows) (light microscope × 400). b. Cultured fungal colonies in case 2 were identified as T.rubrum. c. T. rubrum cultured colonies from case 2. Microconidia  (arrow) were observed under the light microscope, (Medan stained, × 400)

Fig. 3

a. The ventral surface of the nail plate from a normal control. The nail plate was relatively intact without visible damage. (SEM, × 400). b. The ventral surface of the nail plate from case 1. The nail plate was significantly damaged with loose, layered and irregular shape. (SEM, × 500). c. The ventral surface of the nail plate from case 1. Hyphae were piercing through the thin layer of keratinocytes. Keratinocyte layer had scattered bacteria (arrow) (SEM, × 2000). d. The nail plate in case 1 showed significant damage, structural disorder and plenty of hyphae piercing through the thin layer of keratinocytes (arrow). (SEM, × 1000). e. The local amplification of Fig. 3d. There were visible hyphae piercing through the nail plate (arrows). Some of the hyphae were mellow, some were dry, had smooth surface without local destruction, no spores were visible. (SEM, × 3000). f. The image of case 2 showed budding and branching hyphae that piercing through the layered keratinocytes. The hyphae were complete and full. (SEM, × 2000). g. The local amplification of Fig. 3f. There were visible budding and branching hyphae. (SEM, × 5000). h. The image of case 3. There were visible subungual yeast-like cells. (SEM, × 2000). i. The image of case 4. There were subungual hyphae and pseudohyphaes-like features. (SEM, × 4000)

Fig. 4

SEM observation of T. rubrum colonies. Most of the hyphae were straight and smooth, had intact surface without wrinkles. Some of the mycelia were partially dry. Scattered tiny particles were attached to the hyphae surface, had microconidia (arrows) and hyphae branches. (SEM, × 5000)

Fig. 5

a. T. rubrum TEM observation. The image showed the longitudinal, coronal and chamfered surfaces of hyphae and longitudinal section of hyphae branches. The hyphae were constituted by bilateral cell walls, the outer cell wall (OCW) and the inner cell wall (ICW). The longitudinal section of hyphae branches showed visible septal (S), liposomes (L) and lysosomes (Ls). There were high-density particles outside the hyphae. (TEM, × 15000). b. TEM image of the longitudinal section of T. rubrum hyphae showed double-layer cell walls, visible nucleus (N), liposomes (L), the endoplasmic reticulum (ER) and myeloid body (Mb). (TEM, × 50000). c: TEM image of coronal section of T. rubrum hyphae showed double-layer cell walls, the outer cell wall (OCW) and the inner cell wall (ICW), nucleus (N), the nucleolus (Nu), liposomes (L), mitochondria (M) and the endoplasmic reticulum (ER). (TEM, × 70000). d: TEM image of coronal section of T. rubrum hyphae showed mitochondria (M) and ribosomes (G). (TEM, × 100000)