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Home , Mycosis, Onychomycosis

Scanning Electron Microscope Imaging of

William P. Scherer, DPM, MS* Michael David Scherer, BS†

Although scanning electron microscope technology has been used for more than 60 years in many fields of medical research, no studies have focused on obtaining high-resolution microscopic images of onychomy- cosis of the toenail caused by rubrum in a geriatric popu- lation. To provide new insight into the intricate structure and behavior of chronic toenail onychomycosis, we produced three-dimensional images of onychomycosis obtained from two geriatric patients with confirmed growth of T rubrum. The photomicrographs illustrate the pervasive inte- gration and penetration of the hyphal elements, underscoring the clinical difficulty of obtaining rapid treatment of fungal in the distal and lateral subungual space of the human toenail. Although the scanning electron microscope may not be a practical diagnostic tool for most physicians, it remains invaluable for the researcher to obtain insight into the spatial orientation, behavior, and appearance of onycho- . (J Am Podiatr Med Assoc 94(4): 356-362, 2004)

Toenail onychomycosis is the most common condi- mycosis remain effective, they are limited in defining tion diagnosed and treated by podiatric physicians in and detailing the spatial orientation and pervasive- the United States.1 Onychomycosis is usually diag- ness of fungi associated with onychomycosis. Al- nosed using several scientific methods, including though electron microscope technology has been clinical observation, test medium used for more than 60 years in many fields of medi- (DTM) culture, fluorescent potassium hydroxide cal research, no studies have focused on obtaining (KOH) preparation, toenail with surgical high-resolution microscopic images of onychomyco- pathology diagnostic testing, periodic acid–Schiff re- sis of the toenail caused by in action, laboratory fungal culture growth a geriatric population. Rashid et al4 grew fungal cul- analysis, and light microscope imagery.1-3 Conven- tures of Trichophyton mentagrophytes in vitro, find- tional light microscope imaging provides a rapid ing evidence of fungal elements penetrating into the screening test for the presence or absence of fungi to plate. Meyer et al5 investigated infected nails aid physicians when instituting a treatment regimen with T mentagrophytes under the scanning electron for onychomycosis.3 Fungal culture growth by a my- microscope (SEM) and performed a more detailed cology laboratory of specimens obtained by physi- examination of the interaction between the fungus cians can be used to identify the specific genus and and the nail. Our study was undertaken to provide species of particular fungi causing onychomycosis. new insight into the intricate structure and behavior of Although current methods of diagnosing onycho- chronic toenail onychomycosis involving T rubrum. *Private practice, Boca Raton, FL. We obtained samples from the toenails of two geri- †College of Dental Medicine, Nova Southeastern Univer- atric patients suspected of having mycotic sity, Fort Lauderdale, FL. (Research completed during fourth and studied the specimens using mycology laborato- year, University of Miami, Coral Gables, FL.) Corresponding author: Michael David Scherer, BS, PO ry analysis and SEM imaging. Box 272207, Boca Raton, FL 33427 (e-mail: Michael@ Scanning electron microscopes use electrons scherer.net). rather than light to form an image. Electrons have a

356 July/August 2004 • Vol 94 • No 4 • Journal of the American Podiatric Medical Association much smaller wavelength than light, so the SEM can that have an 80% to 90% success rate.12, 13 resolve much smaller structures than the traditional Owing to a lack of published SEM studies of T light microscope. The smallest wavelength of visible rubrum and other mycoses of onychomycosis, a clin- light is approximately 400 nm (400 millionths of a ical sampling of two geriatric patients suspected of meter), whereas the wavelength of electrons used in having fungal infection was conducted for this study. the SEM is approximately 0.005 nm (5 trillionths of a meter). There are many advantages to using the SEM Materials and Methods for medical research instead of a light microscope. The SEM can produce three-dimensional (3-D) im- Two geriatric patients with clinical signs and symp- ages with a large depth of field, which allows a large toms of distal and lateral subungual onychomycosis amount of the sample to be in focus at one time. The were chosen at random from a nursing home setting SEM also produces images of high resolution, which in Fort Lauderdale, Florida. Patient 1 is an 88-year- means that closely spaced features can be examined old woman and patient 2 is an 88-year-old man. Nei- at high magnification (up to ×200,000) and that struc- ther patient had mellitus or was using any tures as small as 2.0 nm can be seen.6 The ability to topical or oral before a speci- produce 3-D medical images with a combination of a men was obtained. Clinical photographs were taken larger depth of field, greater resolution, and higher of each patient’s most affected hallux toenail before magnification than light microscopes makes the SEM specimen collection, and the hallux that appeared to an important diagnostic tool in medical research. have the most clinically infected toenail was chosen for this study. Individual specimens were obtained Clinical Studies by first cleansing the affected toenail and surround- ing skin with a sterile alcohol pad. The infected toe- Onychomycosis is a clinical term used to describe a nail and subungual debris were aggressively debrided fungal infection characterized by thickening, split- as proximally as possible without causing excessive ting, roughening, and discoloration of the toenail.7 discomfort to the patient. Each specimen was ob- People infected with onychomycosis suffer mainly tained from the proximal end of the growing edge of from cosmetic difficulties; however, left untreated, the suspected infection and placed in a small sealed the fungal infection may spread to other toenails and bag (to prevent contamination) labeled with the pa- make everyday activities painful.1 Studies show that tient’s name. fungal infections affect nearly every person who Using sterile technique, both specimens were di- wears shoes at one time or another; darkness, heat, vided into equal halves, with both halves represent- and moisture associated with hosiery and shoes ing the same amount of clinically infected toenail make conditions inviting for the development of ony- and subungual debris. Half of each specimen was left chomycosis.7 The primary fungi associated with ony- in the plastic bag and sent to the Mycology Division chomycosis are T rubrum, T mentagrophytes, and of Quest Diagnostics in Baltimore, Maryland, for flu- spp. The former two fungi are classified as orescent KOH preparation and microscopic fungal —a group of taxonomically related culture examination. The other halves were placed fungi with affinity for cornified , , horns, into small jars containing a 2.5% buffered glutaralde- nails, and feathers.8 Dermatophytes are known to in- hyde solution. The jars were labeled with the pa- fect the ventral nail plate by penetrating lay- tient’s name, stored in a refrigerated container, and ers through enzymatic or mechanical processes.9 transported to the University of Miami Dauer Elec- Trichophyton rubrum is a common dermatophytic tron Microscope Laboratory in Coral Gables, Florida. fungus in southern Florida and throughout the Unit- To preserve the structure of living tissue with no ed States. Approximately 90% of the published cases alteration from the living state, the two toenail sam- of onychomycosis are caused by dermatophytes, ples collected for SEM imaging were fixed in a 2.5% with 90% of these cases caused by T rubrum.10 Fun- glutaraldehyde solution with Millonig’s phosphate gal infections are often spread through contact with buffer (pH = 7.3) for 2 hours at room temperature.6 soils, locker rooms, and bathrooms. Trichophyton After cooling at 4°C overnight, the samples were rubrum is an anthropophilic organism commonly washed three times at 5-min intervals with the same isolated in the laboratory from podiatric medical Millonig’s buffer. The two samples were cross-sec- specimens that are usually present as distal subun- tioned using a -grade razor blade to ob- gual infections (tinea unguium).11 Treatment of ony- tain images of the lateral nail morphology. To add chomycosis can include , topical anti- density and contrast to the biologic tissue by reacting fungal medications, and a regimen of oral antifungal with lipid moieties, postfixation was conducted using

Journal of the American Podiatric Medical Association • Vol 94 • No 4 • July/August 2004 357 a 1% osmium tetroxide solution diluted in buffer for revealed that hyphal elements were seen during the 1.5 hours at room temperature.6 After postfixation, direct fluorescent KOH microscopic examination for the specimens were again washed three times at 5- both patient samples, indicating the presence of der- min intervals with Millonig’s phosphate buffer. In ac- matophytes. In the laboratory, the traditional fluores- cordance with an unpublished procedure for SEM cent KOH preparation method was enhanced by fungal specimen preparation, the samples were dehy- adding the fluorescent dye Calcofluor white, and the drated at room temperature in a graded series of 10% specimen was observed under a fluorescent micro- to 90% ethanol two times at 5-min intervals for each scope.5 After growth on two different types of media step followed by 100% ethanol three times at 10-min —Sabouraud dextrose agar plus chloramphenicol intervals (Dennis Kunkel, PhD, personal communica- and Sabouraud dextrose agar plus chloramphenicol tion, 2003). After the samples were run through the and cycloheximide—for approximately 3 weeks, the dehydration series and contained in absolute ethanol, laboratory microscopically examined the colonies acetone was slowly added to the samples in a 2:1, for genus and species identification. The laboratory 1:1, 2:1, and 100% grade at 10-min intervals. The ace- reported the presence of T rubrum in both samples. tone was gradually replaced with hexamethyldisi- On initial SEM observation of the morphology of lazane in an analogous manner according to the de- the subungual ventral nail plate, we detected fungus hydration protocol to complete critical-point drying hyphal elements in the toenails of the two patients. of the specimens. The dried samples were mounted The thin layers of semihard of the true cuti- ventral side up on aluminum stub mounts using 12- cle, the bed horny layer, and the thicker solehorn mm carbon adhesive tabs coated with carbon-con- provide an environment allowing fungal elements to ducting glue and sputter coated with 6 nm of palladi- 3 um using a Hummer 6.2 sputtering system (Anatach, thrive in patient 1 (Fig. 3). The SEM observation of Ltd, Union City, California). Images were obtained of the cross-sectioned area of the distal toenail in pa- the two patient samples using a Jeol JSM-5600LV SEM tient 1 illustrates fungal hyphae penetrating the ma- (Jeol-USA, Inc, Peabody, Massachusetts) in high-vac- trix between the keratin folds (Fig. 4). Figure 4 fur- uum mode with accelerating voltages at or around ther exemplifies the pervasive nature of T rubrum 2.0 kV. and the difficulty of treatment because of deep pene- tration into the nail plate. Several unique formations Results of hyphae on the toenail plates were also observed; Figure 5 portrays a characteristic heart-shaped de- A complete medical evaluation revealed that patients sign from patient 1. 1 and 2 demonstrated clinical of The SEM observations of the sample obtained distal and lateral subungual onychomycosis (Figs. 1 from patient 2 further illustrate the presence of hy- and 2). Preliminary reports from Quest Diagnostics phae on the distal part of the ventral side of the toe-

Figure 1. Distal and lateral subungual onychomyco- Figure 2. Distal and lateral subungual onychomyco- sis due to Trichophyton rubrum in patient 1. sis due to Trichophyton rubrum in patient 2.

358 July/August 2004 • Vol 94 • No 4 • Journal of the American Podiatric Medical Association 100 µm 10 µm

Figure 3. Distal view of the ventral part of the toenail Figure 4. Lateral view of the cross-sectioned nail exhibiting its unique structure and morphology (×190). shows fungal hyphae integrating between nail plates (×2,500).

20 µm 100 µm

Figure 5. Heart-shaped fungal present on the Figure 6. The ridged ventral nail plate with its tilelike ventral toenail (×600). appearance provides an ideal environment for fungal growth. The white box indicates the location of Tri- chophyton rubrum hyphae (×220).

nail. A fungal hypha is clearly shown in Figure 6 run- ure contains a probable site of keratin digestive ac- ning along the keratin plates of the toenail. Further tivity due to the manner in which the fungus is pene- investigation of the element illustrates the unique trating the nail.14 fungal morphology characteristic of T rubrum (Fig. Throughout the layers of the toenail in patient 2, 7). The average size of the fungal hypha is approxi- arthrospores were seen (Fig. 10); in T rubrum, they mately 2 to 5 µm, with macroaleuriospores reaching are classified as or segments produced by a size of 10 µm. Many of the fungi present on the ven- fragmentation of septate hyphae.8 These elements tral side of the toenail showed evidence of penetra- were isolated to the distal part of the ventral side of tion into the corneocytes; Figure 8 illustrates a hy- the patient’s toenail and appeared to be penetrating phal element protruding from the toenail in patient 2. the corneocytes (Fig. 11). Trichophyton rubrum also Figure 9 presents a magnified view of the area in Fig- tends to reproduce through branching, in which a hy- ure 8 enclosed in a white box. In contrast to the fun- phal element bifurcates on the outermost layer of the gal presence in corneocyte layers in Figure 4, this fig- toenail (Fig. 12).

Journal of the American Podiatric Medical Association • Vol 94 • No 4 • July/August 2004 359 10 µm 20 µm

Figure 7. Magnified view of the framed part of Figure Figure 8. Fungal element suspended above the ven- 6 illustrating the substructure of fungus. The micro- tral side of the nail. The white box indicates a hyphal aleuriospore (I) projects from one of the two projection from the corneocyte (×900). macroaleuriospores (M) (×1,600).

µ 10 µm 20 m

Figure 9. Magnified view of the framed part of Figure Figure 10. Micrograph of the Trichophyton rubrum 8 illustrating penetration of a fungal element into the arthrospore fungal element (×850). corneocyte (×2,300).

Discussion enormous focal depth of the SEM give it a great ad- vantage over the light microscope. Scanning electron microscopy provides valuable in- Our investigation of the samples taken from two sight into the growth characteristics of the fungi as- geriatric patients proved complete in providing posi- sociated with onychomycosis. The SEM allows for tive identification of the fungal elements discovered. far more information about the spatial interaction be- After laboratory genus and species identification, we tween dermatophytes and the nail plate than any proceeded to find hyphae under the SEM. Mycology other medical diagnostic tool. From the study by literature gives insight into the morphologic charac- Meyer et al,5 we ascertained the approximate location teristics of T rubrum. According to the fungus identi- of the fungi and their probable appearance under the fication manual by Funder,14 the elements present in microscope. Although principally investigating T our samples shared the same characteristics as those mentagrophytes, the study by Meyer et al5 concluded listed in his Table 78b. The fluorescent KOH mount that the better resolution, higher magnification, and illustrating growth on the skin is similar to that of

360 July/August 2004 • Vol 94 • No 4 • Journal of the American Podiatric Medical Association 10 µm 10 µm

Figure 11. Magnified view of a fungal arthrospore. An Figure 12. Bifurcating hyphal element present in the arthrospore is a or segment produced by frag- layers of the toenail. Trichophyton rubrum has the mentation of septate hyphae (×1,400). ability to branch off into many different strands of hy- phae (×2,500).

our sample (Figs. 6–9); however, the elements in our The reproductive structures of the fungal infec- samples were mostly isolated fungal hyphae as op- tion were also discovered at the distal part of the posed to structured colonies. The characteristic cigar ventral side of the toenail. On close examination, we shape of the macroconidia, as shown in the manual concluded that the fungi in Figures 10 and 11 were by Funder,14 closely resembles Figure 7 and supports reproducing actively in these locations. Figure E in the laboratory’s identification of the genus and Dermatophytes, by Rebell and Taplin,8 illustrates the species of the dermatophyte infection of the two microscopic appearance of T rubrum arthrospores geriatric patients. Figure 7 illustrates fungal growth and supports the presence of these elements in our characteristics common to T rubrum, including the sample. Dermatophytes, including T rubrum, exhibit usual fluffy thallus consisting of long strands of hy- reduced sporulation in culture and depend on arthro- phae with small, lateral, characteristically tear-shaped spores produced in scalp and desquamating or peg-shaped microaleuriospores.8 human epidermis. Although many of the fungal hyphae were pre- served well in SEM preparation, the elements found Conclusion in the sample from patient 1 had shrunk considerably and compacted the characteristic fungal shape into The SEM allows for greater insight into the intricate one resembling a ribbon (Figs. 4 and 5). The samples structure and behavior of onychomycosis than any from patient 1, however, illustrated the pervasive in- other medical diagnostic tool. By using various tech- tegration of the dermatophyte into the layers of the niques, we obtained high-resolution 3-D images of patient’s nail. As demonstrated in the study by Meyer onychomycosis caused by the fungus T rubrum. Our et al5 of the enzymatic destruction of onychomycosis, photomicrographs illustrate the pervasive integration the SEM allows for visualization of intercellular pen- and penetration of the fungus hyphal elements, un- etration of fungal hyphae into the nail plate between derscoring the clinical difficulty of obtaining rapid the corneocytes. Figure 8 in our investigation sup- treatment of fungal infections in the distal and lateral ports the ability of species in the Trichophyton genus subungual space of the human toenail. The images of to penetrate the nail plate. The fungal element is sus- T rubrum obtained with the SEM show the spatial pended in the toenail and remains well preserved. On orientation and pervasiveness of these fungi associat- closer examination, we observed evidence support- ed with onychomycosis of the toenail with greater ing direct fungal penetration of the corneocytes (Fig. detail than has ever been reported in the medical lit- 9). The hypha, compared with Figure 4, further illus- erature. Although the SEM may not be used by most trates the pervasive integration of the species into physicians treating patients infected with onychomy- the nail plate. cosis, it remains an invaluable tool for the researcher

Journal of the American Podiatric Medical Association • Vol 94 • No 4 • July/August 2004 361 to obtain new insight into the spatial orientation, be- 5. MEYER JC, GRUNDMANN HP, SCHNYDER UW: Onychomyco- havior, and appearance of onychomycosis caused by sis (Trichophyton mentagrophytes): a scanning electron T rubrum. microscopic observation. J Cutan Pathol 8: 342, 1981. 6. BOZZOLA JJ, RUSSELL LD: Electron Microscopy, 2nd Ed, pp 16–18, 154, 204, Jones and Bartlett Publishers, Sud- Acknowledgment. Jeffrey Prince, PhD, of the bury, MA, 1999. University of Miami Dauer Electron Microscope Lab- 7. MCCARTHY DJ: Origins of onychomycosis. Clin Podiatr oratory in Coral Gables, Florida, for his valuable as- Med Surg 12: 221, 1995. sistance with this research project. 8. REBELL G, TAPLIN D: Dermatophytes: Their Recognition and Identification, pp 2, 40–51, 119–121, University of Miami Press, Miami, 1970. References 9. DEBERKER DAR, BARAN R, DAWBER RPR: “Infections In- volving the Nail Unit,” in Handbook of Diseases of the 1. SCHERER WP, KINMON K: Dermatophyte test medium cul- Nails and Their Management, Vol 1, p 53, Blackwell Sci- ture versus mycology laboratory analysis for suspected ence, Oxford, England, 1995. onychomycosis: a study of 100 cases in a geriatric pop- 10. SCHLEFMAN BS: Onychomycosis: a compendium of facts ulation. JAPMA 90: 450, 2000. and a clinical experience. J Foot Ankle Surg 39: 290, 1999. 2. BORKOWSKI P, WILLIAMS M, HOLEWINSKI J, ET AL: Onycho- 11. CRISSEY JT, LANG H, PARISH LC: Manual of Medical My- mycosis: an analysis of 50 cases and comparison of di- cology, p 581, Blackwell Science, Cambridge, England, agnostic techniques. JAPMA 91: 351, 2001. 1995. 3. ELEWSKI BE, CHARIF MA, DANIEL CR III: “Onychomyco- 12. BARAN R, DAWBER RP, TOSTI A, ET AL: A Text Atlas of Nail sis,” in Nails: Therapy, Diagnosis, Surgery, 2nd Ed, ed Disorders, p 165, Martin Dunitz, London, 1996. by RK Scher, CR Daniel, p 151, WB Saunders, Philadel- 13. SCHERER WP, MCCREARY JP, HAYES WW: The diagnosis phia, 1997. of onychomycosis in a geriatric population: a study of 4. RASHID A, SCOTT E, RICHARDSON MD: Early events in the 450 cases in South Florida. JAPMA 91: 456, 2001. invasion of the human nail plate by Trichophyton men- 14. FUNDER S: Practical Mycology, 3rd Ed, p 86, Hafner Pub- tagrophytes. Br J Dermatol 133: 932, 1995. lishing, New York, 1968.

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