INVASION OF NAILS IN VITRO BY CERTAIN DERMATOPHYTES* F. RAUBITSCHEK, M.D. AND RUTH MAOZ Dermatophytes have been grown on natural keratin substrates such as nail fragments and hair (Page (1), Vaa Breuseghem (2, 3), Barlow and Chattaway (4), a.o.). The fungi grow on these substrates in their saprophytie form and no invasion resembling that of the parasitic life phase has been reported. However, it has been shown that when dermatophytes are cultured on a simple medium under conditions of continuous shaking, the parasitic life phase is simulated (5). This technic of growing fungi in shake culture was therefore used in a successful attempt to reproduce parasitic invasion of nails in vitro. METHODS AND MATERIALS The medium consisted of 0.3 per cent ammonium sulfate, 0.2 per cent dibasic potassium phosphate, 0.01 per cent soluble yeast extract (Difco), 0.005 per cent magnesium-sulfate and 0.005 per cent calcium chloride in double-distilled water. The medium was dispensed in 100 ml portions into 250 ml Erlenmcyer flasks and autoclaved at 15 atmospheric pressure for 15 mm. The inoculum was prepared as reported previously (5) from freshly isolated strains. Two strains each of T. mentagroph pica and T. rubrum and one strain of T. tonsurens were tested. Finger nail clippings from 3 adults and 2 children previously found free from fungi when examined in KOH preparations were used. The clippings were washed for 6 consecutive days under running tap water and for 24 hours in several changes of distilled water, then in 3 hourly changes of 96 per cent ethanol and finally in 2 brief changes of ether. Subsequently they were dried in sterile Petri dishes at 37 C. Five different experiments were set up simultaneously. About 10 pieces of nail and 0.2 ml of inoculum were added to a flask of medium and to a flask containing distilled water only. To another flask of medium and to one containing distilled water only, 0.2 ml of inoculum were added. To another flask containing medium only, 10 pieces of nail were added without inoculum. In each series 3 flasks were set up and incubated at 28 C on a continuous shaking machine (Ross-Kershaw) at 148 strokes per minute for 2 4 weeks. At various intervals pieces of nails, pellets of fungal growth and 10 ml. portions of culture fluid were withdrawn aseptically. The nails were examined microscopically in KOH preparations, put on glucose agar slants and processed histologically after fixation in formol or trichloracctic acid-ethanol. The sections were made accordiug to the method of Sagher (6). Some sections were stained with hematoxylin-cosin (H and E) and the rest with periodic acid Schiff stain (PAS). It was apparent from the start that the sections stained with H and E were inferior by far to those stained with PAS and showed almost no clearly stained fungal elements. Therefore all observations were made on PAS stained sections only. In addition, histochemical stains for SS and SR (7, 8) groups were employed. The pellets of fungal growth were examined microscopically and grown on glucose agar slantstoverifyspecificityand sporulation. Thecultureliquid wasmeasured with a Beckman glass electrode ph meter, its total nitrogen content determined by the Conway (9) method after boiling with concentrated sulfuric acid and qualitative tests for amino acids (10, SS and SR groups were made (11, 12). * From the Department of Dermatology and Venereology (A. Dostrovsky, Director), Hebrew University-Hadassah Medical School, Jerusalem, Israel. Received for publication July 25, 1956. 261
262 THE JOURNAL OF INVESTIGATIVE I)ERMATOLOGY -a-,;'. y FIG. 1. Early invasion of nail by T. tonsurans during continuous shaking for 6 days. Magnification X 560. These and all subsequent sections stained with PAS. 4, / FIG. 2. Early invasion of nail plate by T. rubrum during continuous shaking for 7 days. Note hyphae pushing forward between laminae of nail. Magnification X 560.
INVASION OF NAILS BY BEIIMATOPHYTES 263 I FIG. 3. Heavy invasion of nail plate by T. rubrnm dnring continuous shaking for 14 days. Arthrospore formation and splitting of laminar structure of nail. Magnification X 560. FIG. 4. Invasion of nail plate by T. mentagrophytes from free edge of nail clipping. Note numerous hyphae entering nail plate parallel to the laminae. The fungal growth separates the laminae leading to formation of cracks and slits. Magnification X 560.
264 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY a- Fin. 5. Heavy invasion of nail plate by T. mentagrophytes during continuous shaking for 20 days. Note arthrosporc formation and slits in nail plate. Magnification X 560. I -r Fin. 6. Invasion of nail plate by T. mentagropbytes during continuous shaking for 14 days. Note hyphal character of fungal growth on nail surface. Magnification X 160.
INVASION OF NAILS BY DERMATOPHYTES 265 Fins. 7, 8, 9. Control. Nail clippings after various periods of shaking. Note absence of lits or cracks. Fig. 7: Magnification X 150; Fig. 8: Magnification X 370; Fig. 9: Magnificainn X 560. a -,.-,- - -'---.. '.._4 dt?-. - -.jt..' - - -.-.k -. -a Fin. 8
266 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY - '- Fio. 9 RESULTS The flasks with medium containing oniy fungi showed pellets of fungal growth after 5 to 6 days of shaking. The flasks containing distilled water and fungi did not show any fungal growth at any time. The flasks containing medium, fungi and nails showed no fungal pellets but the pieces of nail were covered with a short white fuzzy growth. This superficial growth, which was absent when the medium was replaced by distilled water (only one strain of T. mentagrophytes was used in the experiments in which the medium was replaced by distilled water) consisted microscopically of a dense maze of hyphae with occasional arthrospore formation. Here and there single hyphae were seen entering the nail plate through breaks in the surface. In later stages the surface mat was denser and hyphae which had invaded the nail were seen running roughly parallel to the nail surface in slits and cracks between the laminae. Some of these hyphae showed arthrospore formation. No hyphae were seen growing perpendicular to the laminar structure of the nails. The nail sections from the flasks in which the medium was replaced by distilled water also showed this type of invasion, but there was no fungal growth covering the surface of these pieces of nail. Glucose agar slants seeded with parasitized pieces of nails yielded typical granular colonies of the respective invading organism. The sections of nails from the control flasks not inoculated with fungi showed almost absolute absence of cracks and slits and no fungal growth. Cultures from these nails on glucose agar slants remained sterile. The special stains for SS and S-H groups did not show proof of keratolysis.
INVASION OF NAILS BY DERMATOPHYTE5 267 The culture fluid of all series was always negative for free amino acids and SS and SH groups. The nitrogen determinations showed no significant differences in the various experiments. COMMENT The systematic histological study of fungus infected nails was first undertaken by Sagher (6). The PAS stain was not in use for fungi at that time and all his observations were done on sections stained with H and E. He observed fungal hyphae lying parallel to the nail laminae apparently pushing them apart. In the above experiments the parasitic type of invasion of nail clippings could be produced in vitro and the same phenomenon was observed. Since almost no cracks or slits were found in the nails from flasks to which no fungus inoculum had been added, it appears probable that the hyphac, after having gained a foothold in surface breaks of the nails, penetrated between the laminae and pushed them apart. No evidence for keratolysis, enzymatic or otherwise, was found in these experiments. It would appear therefore that the damage to the nail caused by the invading fungus was due to mechanical rather than chemical action. Though the composition of the medium is sufficient for at least minimal amount of growth of the fungi, distilled water is definitely insufficient. Apparently the organisms utilize non-keratin compounds in nail as found by Bolliger and Gross (13). SUMMARY 1. Invasion of nail clippings by certain dermatophytes was induced in vitro by the use of the continuous shake culture technic. 2. In sections of parasitized nail clippings stained by the PAS technic, hyphac with occasional arthrospore formation were seen growing between the laminae of the nail, apparently pnshing them apart. No evidence of keratolysis was found and the damage to the nail prodnced by the fungi seemed to be due to mechanical rather than chemical action. REFERENCES 1. PAGE, H. M.: Observations on keratin digestion by M. gypseum. Mycologia, 42: 591 602, 1950. 2. VAN BEEUSEGHEM, H.: La cnlture de dermatophytes in vitro sur des cheveuz isolés. Ann de parasitol., 24: 559-573, 1949. 3. VAN BREU5EGHEM, H.: Keratindigestion by dermatophytes: A specific diagnostic method. Mycologia, 44: 176 182, 1952. 4. BAELOW, A. J. E. AND CHATTAWAY, F. W.: The attack of chemically modified keratin by certain dermatophytes. J. Invest. Dermat., 24: 65 74, 1955. 5. RAIJBITsCHEK, F.: Nutritional requirements of dermatophytes in continuous shake culture. J. Invest. Dermat., 25: 83 87, 1955. 6. SAGHER, F.: Histologic ezaminations of fungous infections of the nails. J. Invest. Dermat., 11: 337 354, 1948. 7. CHEvEEMONT, M. AND FREDERIC, J.: Une nouvelle methode histochimique de misc en evidence des substances a fonction sulfhydrilc Arch de biol., Paris, 54: 589 605, 1943. S. GOLDBLUM, H. W., PIPER, W. N. AND CAMPBELL, A. W.: A comparison of three histochemical stains for the demonstration of protein bound sulfhydril groups in normal human skin. J. Invest. Dcrmat., 23: 375 383, 1954.
268 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 9. CONWAY, B. J. AND BYRNE, A.: The microdetermination of ammonia. Bioehem. J., 27: 419 429, 1933. 10. FmoL, F.: Spottests, Vol. II., 209. NeW York, Elsevier Pubi. Comp., 1954. 11. GRUNERT, II. It. AND PHILLIPS, II. H.: A modification of the nitroprusside method of analysis for glutathion. Arch. Biochem., 30: 217 225, 1951. 12. AVIDOR, Y. AND MAGER, J.: A spectrophotometric method for determination of cystein and related compounds. J. Biol. Chem., 1956 (in press). 13. BOLLIGER, A. AND GRoss, It.: Non keratins of human toe nails. Anstralian J. Exper. Biol. & M. Sc., 31: 127 130, 1953.