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Pityrosporum Orbiculare
VOL. 63, NO. 4
LATERAL VERSION LIGHT-REFLEX TEST
results were best and most stable when sur gery normalized the LVR. The LVR findings were helpful in pre dicting whether a postoperative over- or undercorrection would improve or would re quire further surgery. 412 Phoenix Street ,(49090)
815
REFERENCES
1. Burian, H. M. : Principles of surgery on the extraocular muscles. Am. J. Ophth. 33:380, 19S0. 2. Guibor, G. : Squint and Allied Conditions. New York, Grune & Stratton, 1959, p. 28. 3. Lyle, T. K. : Worth and 'Chavasses's Squint, Philadelphia, Blakiston, 1950, p. 208.
P I T Y R O S P O R U M ORBICULARE ITS POSSIBLE ROLE IN SEBORRHEIC BLEPHARITIS ALEKSANDAR PARUNOVIC, M.D.,
AND CARLYN HALDE, P H . D .
San Francisco, California The possibility that the genus Pityrosporum might play an etiologic role in sebor rheic blepharitis was suggested by Thygeson 1 in 1946. He had noticed that yeastlike orga nisms occurred more commonly on the lid margins of patients suffering from seborrheic blepharitis, or from mixed seborrheic-staphylococcic blepharitis, than on the lid margins of patients with pure staphylococcic infec tion. Rivolta, in 1873, and Malassez,2 in 1874, were the first to describe the budding yeasts found on seborrheic skin. Later Unna studied the organism more closely, and it was known as "the bottle bacillus of Unna" until Sabouraud gave it the generic name Pityrosporum and called the species P. ovale. Its fastidious nature made isolation and experimental study difficult until Meirowsky, Templeton and, later, Benham3 discovered that it needed fatty substances From the Francis I. Proctor Foundation for Research in Ophthalmology and the Department of Microbiology, University of California San Francisco Medical Center. This investigation was conducted under a Fight for Sight Postdoctoral Research Fellowship (F-188) of the National Council to 'Combat Blindness, Inc., New York, and supported in part by United States Public Health Service research grants NB 06207 and NB 00604.
for growth. Even now, isolation attempts are not always successful. Study of the relationship of Pityrospo rum to seborrheic disease was intensified between 1935 and 1955. Moore4 and Kile and Engman 5 felt that Pityrosporum was the cause of seborrhea, and Moore support ed his statement with experimental studies. Later, however, in the course of extensive clinical and experimental investigations, Martin-Scott 6 and Spoor and his group 7 iso lated P. ovale from normal skin as often as from seborrheic skin. Martin-Scott's human inoculations did not result in seborrhea, and his attempts to induce hypersensitivity and antibody formation in animals were unsuc cessful. In the recent dermatologie litera ture, P. ovale is rarely mentioned and has been discarded as a possible cause of seborrhea.8 In the ophthalmologic literature, Gotts and Thygeson9 reported isolating P. ovale from lid margins in 1947. François 10 re ported two cases of severe blepharoconjunctivitis which he believed to be caused by P. ovale. Vidal 11 felt that these yeasts were definitely the cause of seborrheic blepharitis but did not support his statement with clini cal or laboratory evidence. In Duke-Elder's1* opinion, P. ovale might be a contributing
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Fig. 1 (Parunovic and Halde). Scale from seborrheic lid margin, showing abundant P. orbiculare (methylene-blue stain, χΙΟΟΟ).
factor. It is significant that the ophthalmologic observers (Thygeson, 9 Feducowitz,13 François, 10 Vidal 11 ) all described the orga nisms seen on the lid margin as predomi nantly spherical rather than bottle-shaped. In 1951, a spherical lipophilic yeast, differing from the oval, bottle-shaped P. ovale, was isolated by Gordon14 from scales of tinea versicolor and was named P. orbi culare. Of Gordon's 15 isolations, it is inter esting that two were from normal skin. Some authors 15 ascribe pathogenicity to P. orbiculare, regarding it as the possible cause of tinea versicolor. The present study was undertaken in an effort to evaluate the role of Pityrosporum in seborrheic blepharitis by three approach es: (1) cultural study of lipophilic orga nisms found on normal and seborrheic lid margins; (2) microscopic examination of scrapings from normal and seborrheic lid margins, and from related areas, with a comparison of the frequency of occurrence, the number, and the types of Pityrosporum seen; and (3) attempts to produce sebor rheic lesions in experimental animals with an isolate of P. orbiculare. 1. L I D MARGIN CULTURES MATERIALS AND METHODS
Material for culture was obtained by col lecting scales from the lid margins (and in parallel attempts from the scalp, forehead, and eyebrows) of 50 patients with sebor
rheic blepharitis and 20 patients with nor mal lid margins. The lid margins and other areas were scraped with a platinum spatula and the scales collected with a small forceps. The medium chosen for primary isolation was Sabouraud's dextrose agar, fortified with Actidione to prevent growth of saprophytic fungi and with chloramphenicol to prevent growth of bacteria. After inocula tion, the medium was overlaid with sterile olive oil and incubated at 37°C. It was soon obvious that chloramphenicol was not suffi cient to prevent the growth of staphylococci, probably because most of our patients had previously been treated with antibiotics and were harboring chloramphenicol-resistant staphylococci. Thenceforth, the scales were washed in methicillin solution before being inoculated onto the media. This small change resulted in more frequent isolations. RESULTS
From the 50 patients with seborrheic ble pharitis, 18 isolations of Pityrosporum were obtained; seven were P. orbiculare, three were P. ovale, and seven were a mixture of the two. Parallel cultures from scalp, fore head and eyebrows from these same patients yielded only P. ovale, with the exception of one mixed culture. It was concluded that P. orbiculare is more often harbored on the lid margin than on the facial skin or scalp of seborrheic patients. From the lid margins of the 20 persons
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having no signs of seborrheic blepharitis, seven isolates were obtained; two were P . orbiculare, two were P. ovale, and three were a mixture of the two. It appears, therefore, that P. orbiculare is not limited to the seborrheic lid margin since it was isolat ed also and in roughly the same proportion of cases, from apparently normal lid mar gins. BIOLOGIC PROPERTIES OF P. ORBICULARE
P. orbiculare is even more fastidious in its growth requirements than P. ovale. It either does not grow or grows very poorly in oleic acid or on wort agar, the two media mostly commonly used for the isolation of P. ovale. Since one of Gordon's strains formed hyphae on corn-meal agar at 30°C,16 we repeated this experiment. None of our isolates formed hyphae on this medium but one strain formed short hyphae on carrot overlaid with olive oil. When P. orbiculare was stained with méthylène blue, Giemsa stain, Gram stain, PAS and the oil-red technique, méthylène blue was found to be the best stain for scrapings and PAS the best for morpho logic studies. Although the organism was generally gram-positive, an occasional cell
Fig. 2 (Parunovic and Halde). Scale from se borrheic scalp, showing P. ovale (methylene-blue, X1000).
Fig. 3 (Parunovic and Halde). P. orbiculare, culture (PAS stain, χ1480).
was gram-negative. This staining variation has been described by Keddie.15 The result of staining for lipids deserves special comment. The center of the spherical fungal cell, which by other staining tech niques appears to be a vacuole, stained with oil-red. This confirmed the conclusion of Barfatani, 17 who studied P. orbiculare with the electron microscope, that the "vacuole" is probably a lipid globule. The morphology, color, and characteristic odor of the colonies were the same as de scribed in the literature. Martin-Scott found P. ovale very resistant to all antibiotics tested and to many inor ganic compounds, including phenol. Spoor18 tested the germicidal activity of Selsun sus pension and found it active against P. ovale. He used a cup-plate assay method, however, overlooking the fact that selenium sulfide is insoluble in water and thus could not diffuse into the medium. In our study, we tested Selsun ophthalmic ointment by adding it to the olive oil used to overlay inoculated plates; no growth of either P. ovale or P. orbiculare was observed. Our parallel cultures have shown that, for
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AMERICAN JOURNAL OF OPHTHALMOLOGY
reasons unknown, P. orbiculare can be iso lated with greater frequency from the lid margins than from the facial skin or scalp of the same patient. It may be that these spherical organisms find some specific nu trient more abundant in secretions from the meibomian and zeisian glands than on other skin surfaces. Very little is known about these secretions. Zeiss's glands were consid ered by Duke-Elder19 to be similar to the meibomian glands, and by Wolf20 to be sim ply degenerated sebaceous glands. Recently Linton 21 and others have shown that the secretion of the meibomian glands is unique in that it contains no fatty acids or cholesterol, which are the important prod ucts of all other sebaceous glands. Ac cording to their study, meibomian glands secrete an unidentified lipid that cannot be found in the secretion of sebaceous glands of the skin. This could perhaps be one of the factors determining the abundance of P. orbiculare on the lid margins. 2. MICROSCOPIC EXAMINATION OF SCRAPINGS BUDDING YEASTS IN LID MARGIN SCRAPINGS
Lid margin scrapings are often of great diagnostic value in external diseases of the eye. It was attempted in this study to deter mine whether or not the presence of bud ding yeastlike organisms could be helpful in the diagnosis of blepharitis. Portnoy 22 examined 250 scrapings from the scalp and found P. ovale on 100% of seborrheic and 85% of nonseborrheic scalps. Thygeson and Vaughan 23 found yeastlike organisms on the lid margins of 98%' of pa tients with seborrheic blepharitis and 58% of patients with normal lid margins. In our series of 50 patients with sebor rheic blepharitis, microscopic examination revealed budding yeasts in all scrapings but with a considerable difference in the shape and number of organisms seen. Spherical forms only, or spherical forms mixed with a few oval forms, were seen in 42 scrapings,
APRIL, 1967
oval forms alone in only eight. When scrap ings from 87 patients with normal lid mar gins were examined, spherical forms only or predominantly were seen in scrapings from 81 patients (93%), and no budding yeast like forms were seen in scrapings from the other six patients ( 7 % ) . If, however, we compared the number of organisms seen on seborrheic lid margins with the number seen on normal lid mar gins, yeasts were more numerous on the se borrheic lid margin than on the normal lid margin. But this finding was neither consis tent nor diagnositc. As far as a diagnosis of blepharitis was concerned, the spherical yeast, P. orbiculare, was found in sig nificantly greater numbers only in cases of clinically obvious seborrheic disease. In borderline cases or mild cases, the number of organisms present on the lid margins was of no help in determining etiology. Three of our patients showed very marked scaling, but their lid margins were not inflamed and they had no complaints; P. orbiculare was seen in great numbers in their scrapings and was isolated in culture. Contrary to this, a few patients with clinical evidence of sebor rheic blepharitis, including marked lid mar gin irritation, showed only a few organisms in their lid-margin scrapings, and no isola tions were obtained in culture. SEARCH FOR BUDDING YEASTS AT SPECIAL SITES
1. At the mucocutaneous junction. Vidal claims to have found spherical yeasts in 100% of scrapings from the intramarginal space, that is, between the anterior and pos terior edges of the lid. In an effort to evalu ate his findings, we prepared scrapings from the same area but were either unable to find Pityrosporum or found only a few. 2. In meibomian gland channels. If P. orbiculare lives on the lid margins because of a specific secretion of the meibomian glands, one would expect the yeast to be present in meibomian gland channels. To investigate this possibility, tissues were col-
' VOL. 63, NO. 4
PITYROSPORUM ORBICULARE
lected from the upper tarsal plate of 20 ca davers, three of which had scaly lid mar gins. Histologie sections of these tissues were stained with Gridley stain but no fun gus elements were seen in the gland chan nels. 3. In hair shaft follicles. Spherical yeasts were found surrounding the hair shaft, low in the follicle, when eyelashes were epilated. 3. ANIMAL INOCULATION EXPERIMENTS
Experimental inoculations with either P. ovale or P. orbiculare have failed to produce clinical evidence of seborrhea except under unusual conditions. For example, Moore and his associates5 inoculated a group of seborrheic individuals with P. ovale and re ported 40% positive results. When the ob jection was raised that Moore was produc ing seborrhea in seborrheic individuals, Martin-Scott 6 repeated the experiment with 51 normal, nonseborrheic volunteers and was unable to produce seborrhea. Other at tempts have been made, most of them by an imal inoculation, which have been either un successful or disputable.6·7'9'10'22·24 MATERIALS AND METHODS
In our experiments the inoculum was a pure culture of P. orbiculare isolated from a patient with severe seborrheic blepharitis. Fifteen rabbits were selected as experimental animals. Prior to selection, all animals were examined with the slitlamp. Some of them, interestingly, had fine scales on their lid margins. These were not used for the study but scales from their lid margins were stained with méthylène blue for direct mi croscopic examination and were cultured as already described. No yeastlike organisms were seen or isolated. The rabbits to be inoculated were divided into three groups : Group 1. Six animals were inoculated by rubbing a four-day-old culture on the lower lid of the right eye and on a small shaved area of the back. The sites of inoculation were superficially scarified because previous
819
reports stated that a positive result could not be expected unless this was done. The left lid margin was inoculated in the same way except that Saccharomyces cerevisiae, a known nonpathogenic yeast, was used as a control. The culture material was applied both to the skin and to the lid margin once a day for four days. Group 2. Four rabbits received a suspen sion of live P. orbiculare in the conjunctival sac of the right eye, and the control fungus in the conjunctival sac of the left eye, twice a day for two weeks. Group 3. Four rabbits received twice a day for two weeks a cell extract of P. orbic ulare prepared according to the method used to prepare Trichophytin. 25 As a con trol, a fifth animal was given once a day a drop of the medium used for growing P. orbiculare. RESULTS
Material from the lid margins of the rab bits in the first group was collected for mi croscopic examination and culture on the fifth, 12th and 21st days after inoculation. On the fifth day, all scrapings and cultures were positive. On the 12th day, organisms were seen in all scrapings but only two cul tures were positive for P. orbiculare. Final ly, on the 21st day, only a few spherical forms were seen in scrapings from the lid margins of two rabbits; all other scrapings and cultures were negative. Saccharomyces were seen in the scrapings made on the fifth and 12th days. During 30 days of observation, no significant clinical change was observed. There was some scaliness during the first week, but similar changes were also ob served in the controls. The animals in the second and third groups also showed no clinical change dur ing 30 days of observation. SUMMARY AND CONCLUSIONS
1. In an attempt to evaluate the role of the lipophilic yeasts in the etiology of sebor-
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6. Martin-Scott, J. : The Pityrosporum ovale. Brit. J. Dermat. 64:2S7, 1952. 7. Spoor, H. J., Traub, E. F. and Bell, M.: Pi tyrosporum types cultured from normal and se borrheic subjects. Arch. Derm. & Syph. 69:323, 1954. 8. Pillsbury, D. M., Shelley, W. B. and Klingman, A. M. : Textbook of Dermatology. Philadel phia, Saunders, 1956, p. 120. 9. Gotts, J. S., Thygeson, P. and Waisman, M. : Observations on P. ovale in seborrheic blepharitis and conjunctivitis. Am. J. Ophth. 30:1485, 1947. 10. François, J. and Rabley, M. : Etude cytologique et bactériologique des frottis conj. de P. ovale. Ann. Ocul. 183:378, 1950. 11. Vidal, F . : Zeisitis caused by the round Pi tyrosporum. Arch. Oftal. B. Aires, 27 :358, 1952. 12. Duke-Elder, S. : System of Ophthalmol ogy : Vol. Vili. London, Compton, 1965, pp. 397, 546. 13. Feducowitz, H. B. : External infections of the eye. Appleton, New York, 1963, p. 186. 14. Gordon, M. A. : The lipophilic microflora 4. Rabbits were inoculated with both liv of the skin. Mycologia, 43 :524, 1951. ing organisms and cell extracts of P. orbicu 15. Keddie, F. and Shadomy, S. : Etiological lare. Thirty days of observation failed to significance of P. orbiculare in tinea versicolor. Sabouraudia, 3:21, 1963. reveal any significant lesions or changes in 16. Gordon, M. A. : Lipophilic yeast like orga the infected animals. nisms associated with tinea versicolor. Invest. 5. No convincing proof of anything more Derm. 17 :267, 1951. 17. Barfatani, M., Munn, R. and Schjeilde, O. than a casual relationship between Pityro- A. : An ultrastructure study of Pityrosporum or sporum and seborrheic blepharitis could be biculare. J. Invest. Derm. 43 :231, 1964. 18. Spoor, H. J. : A study of antidandruff established. agents. Drug & Cosm. Ind. 77:44, 1955. San Francisco Medical Center (94122) 19. Duke-Elder, S. : System of Ophthalmol ogy: Vol. II. London, Compton, 1961, p. 526. REFERENCES 20. Wolf, E. : Anatomy of the Eye and Orbit. 1. Thygeson, P. : Etiology and treatment of London, Lewis, 1961, ed. 5, p. 173. blepharitis. Arch. Ophth. 36:44S, 1946. 21. Linton, R. G., Curnow, D. H. and Riley, W. 2. Malassez, L. : Note sur le champignon du J. : The Meibomian glands. Brit. J. Ophth. pityriasis simple. Arch. Physiol. 2:4S1, 1874. 45:718, 1961. 3. Benham, R. W. : The cultural characteristics of 22. Portnoy, B. : Seborrheic dermatitis. Med. P. ovale : · A. lipophilic fungus. J. Invest. Derm. 111.4:1,1950. 2:187, 1939. 23. Thygeson, P. and Vaughan, D. G. : Sebor 4. Moore, M. : Cultivation and study of P. rheic blepharitis. Tr. Am. Ophth. Soc. 52:173, ovale, the so-called bottle bacillus of Unna. Arch. 1954. Derm. & Syph. 31:661 1935. 24. Emmons, C. W. : The isolation and pathogen5. Moore, M., Kile, R. M. and Engman, M. F . : icity of P. ovale. Pub. Health Rep. 55:1306, 1940. P. ovale, cultivation and possible role in sebor 25. Muskatblit, E. and Director, W. : The Trichorheic dermatitis. Arch. Derm. & Syph. 33:457, 1936. phytin test. Arch. Derm. & Syph. 27:739, 1933. rheic blepharitis, P. orbiculare Gordon was isolated from the lid margins of 14 seborrheic and five nonseborrheic individuals, ei ther in pure culture or mixed with P. ovale. 2. Parallel cultures and scrapings from the glabrous facial skin and scalp of these same patients showed very few or no P. or biculare. It was concluded that P. orbiculare is more common on the lid margins than on the facial skin or scalp. 3. Microscopic studies of lid margin scrapings showed that P. orbiculare is har bored by as many individuals with normal lid margins as with seborrheic lid margins. It is unlikely, therefore, that P. orbiculare bears a causal relationship to seborrheic blepharitis.
LATERAL VERSION LIGHT-REFLEX TEST
results were best and most stable when sur gery normalized the LVR. The LVR findings were helpful in pre dicting whether a postoperative over- or undercorrection would improve or would re quire further surgery. 412 Phoenix Street ,(49090)
815
REFERENCES
1. Burian, H. M. : Principles of surgery on the extraocular muscles. Am. J. Ophth. 33:380, 19S0. 2. Guibor, G. : Squint and Allied Conditions. New York, Grune & Stratton, 1959, p. 28. 3. Lyle, T. K. : Worth and 'Chavasses's Squint, Philadelphia, Blakiston, 1950, p. 208.
P I T Y R O S P O R U M ORBICULARE ITS POSSIBLE ROLE IN SEBORRHEIC BLEPHARITIS ALEKSANDAR PARUNOVIC, M.D.,
AND CARLYN HALDE, P H . D .
San Francisco, California The possibility that the genus Pityrosporum might play an etiologic role in sebor rheic blepharitis was suggested by Thygeson 1 in 1946. He had noticed that yeastlike orga nisms occurred more commonly on the lid margins of patients suffering from seborrheic blepharitis, or from mixed seborrheic-staphylococcic blepharitis, than on the lid margins of patients with pure staphylococcic infec tion. Rivolta, in 1873, and Malassez,2 in 1874, were the first to describe the budding yeasts found on seborrheic skin. Later Unna studied the organism more closely, and it was known as "the bottle bacillus of Unna" until Sabouraud gave it the generic name Pityrosporum and called the species P. ovale. Its fastidious nature made isolation and experimental study difficult until Meirowsky, Templeton and, later, Benham3 discovered that it needed fatty substances From the Francis I. Proctor Foundation for Research in Ophthalmology and the Department of Microbiology, University of California San Francisco Medical Center. This investigation was conducted under a Fight for Sight Postdoctoral Research Fellowship (F-188) of the National Council to 'Combat Blindness, Inc., New York, and supported in part by United States Public Health Service research grants NB 06207 and NB 00604.
for growth. Even now, isolation attempts are not always successful. Study of the relationship of Pityrospo rum to seborrheic disease was intensified between 1935 and 1955. Moore4 and Kile and Engman 5 felt that Pityrosporum was the cause of seborrhea, and Moore support ed his statement with experimental studies. Later, however, in the course of extensive clinical and experimental investigations, Martin-Scott 6 and Spoor and his group 7 iso lated P. ovale from normal skin as often as from seborrheic skin. Martin-Scott's human inoculations did not result in seborrhea, and his attempts to induce hypersensitivity and antibody formation in animals were unsuc cessful. In the recent dermatologie litera ture, P. ovale is rarely mentioned and has been discarded as a possible cause of seborrhea.8 In the ophthalmologic literature, Gotts and Thygeson9 reported isolating P. ovale from lid margins in 1947. François 10 re ported two cases of severe blepharoconjunctivitis which he believed to be caused by P. ovale. Vidal 11 felt that these yeasts were definitely the cause of seborrheic blepharitis but did not support his statement with clini cal or laboratory evidence. In Duke-Elder's1* opinion, P. ovale might be a contributing
816
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APRIL, 1967
Fig. 1 (Parunovic and Halde). Scale from seborrheic lid margin, showing abundant P. orbiculare (methylene-blue stain, χΙΟΟΟ).
factor. It is significant that the ophthalmologic observers (Thygeson, 9 Feducowitz,13 François, 10 Vidal 11 ) all described the orga nisms seen on the lid margin as predomi nantly spherical rather than bottle-shaped. In 1951, a spherical lipophilic yeast, differing from the oval, bottle-shaped P. ovale, was isolated by Gordon14 from scales of tinea versicolor and was named P. orbi culare. Of Gordon's 15 isolations, it is inter esting that two were from normal skin. Some authors 15 ascribe pathogenicity to P. orbiculare, regarding it as the possible cause of tinea versicolor. The present study was undertaken in an effort to evaluate the role of Pityrosporum in seborrheic blepharitis by three approach es: (1) cultural study of lipophilic orga nisms found on normal and seborrheic lid margins; (2) microscopic examination of scrapings from normal and seborrheic lid margins, and from related areas, with a comparison of the frequency of occurrence, the number, and the types of Pityrosporum seen; and (3) attempts to produce sebor rheic lesions in experimental animals with an isolate of P. orbiculare. 1. L I D MARGIN CULTURES MATERIALS AND METHODS
Material for culture was obtained by col lecting scales from the lid margins (and in parallel attempts from the scalp, forehead, and eyebrows) of 50 patients with sebor
rheic blepharitis and 20 patients with nor mal lid margins. The lid margins and other areas were scraped with a platinum spatula and the scales collected with a small forceps. The medium chosen for primary isolation was Sabouraud's dextrose agar, fortified with Actidione to prevent growth of saprophytic fungi and with chloramphenicol to prevent growth of bacteria. After inocula tion, the medium was overlaid with sterile olive oil and incubated at 37°C. It was soon obvious that chloramphenicol was not suffi cient to prevent the growth of staphylococci, probably because most of our patients had previously been treated with antibiotics and were harboring chloramphenicol-resistant staphylococci. Thenceforth, the scales were washed in methicillin solution before being inoculated onto the media. This small change resulted in more frequent isolations. RESULTS
From the 50 patients with seborrheic ble pharitis, 18 isolations of Pityrosporum were obtained; seven were P. orbiculare, three were P. ovale, and seven were a mixture of the two. Parallel cultures from scalp, fore head and eyebrows from these same patients yielded only P. ovale, with the exception of one mixed culture. It was concluded that P. orbiculare is more often harbored on the lid margin than on the facial skin or scalp of seborrheic patients. From the lid margins of the 20 persons
VOL. 63, NO. 4
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having no signs of seborrheic blepharitis, seven isolates were obtained; two were P . orbiculare, two were P. ovale, and three were a mixture of the two. It appears, therefore, that P. orbiculare is not limited to the seborrheic lid margin since it was isolat ed also and in roughly the same proportion of cases, from apparently normal lid mar gins. BIOLOGIC PROPERTIES OF P. ORBICULARE
P. orbiculare is even more fastidious in its growth requirements than P. ovale. It either does not grow or grows very poorly in oleic acid or on wort agar, the two media mostly commonly used for the isolation of P. ovale. Since one of Gordon's strains formed hyphae on corn-meal agar at 30°C,16 we repeated this experiment. None of our isolates formed hyphae on this medium but one strain formed short hyphae on carrot overlaid with olive oil. When P. orbiculare was stained with méthylène blue, Giemsa stain, Gram stain, PAS and the oil-red technique, méthylène blue was found to be the best stain for scrapings and PAS the best for morpho logic studies. Although the organism was generally gram-positive, an occasional cell
Fig. 2 (Parunovic and Halde). Scale from se borrheic scalp, showing P. ovale (methylene-blue, X1000).
Fig. 3 (Parunovic and Halde). P. orbiculare, culture (PAS stain, χ1480).
was gram-negative. This staining variation has been described by Keddie.15 The result of staining for lipids deserves special comment. The center of the spherical fungal cell, which by other staining tech niques appears to be a vacuole, stained with oil-red. This confirmed the conclusion of Barfatani, 17 who studied P. orbiculare with the electron microscope, that the "vacuole" is probably a lipid globule. The morphology, color, and characteristic odor of the colonies were the same as de scribed in the literature. Martin-Scott found P. ovale very resistant to all antibiotics tested and to many inor ganic compounds, including phenol. Spoor18 tested the germicidal activity of Selsun sus pension and found it active against P. ovale. He used a cup-plate assay method, however, overlooking the fact that selenium sulfide is insoluble in water and thus could not diffuse into the medium. In our study, we tested Selsun ophthalmic ointment by adding it to the olive oil used to overlay inoculated plates; no growth of either P. ovale or P. orbiculare was observed. Our parallel cultures have shown that, for
818
AMERICAN JOURNAL OF OPHTHALMOLOGY
reasons unknown, P. orbiculare can be iso lated with greater frequency from the lid margins than from the facial skin or scalp of the same patient. It may be that these spherical organisms find some specific nu trient more abundant in secretions from the meibomian and zeisian glands than on other skin surfaces. Very little is known about these secretions. Zeiss's glands were consid ered by Duke-Elder19 to be similar to the meibomian glands, and by Wolf20 to be sim ply degenerated sebaceous glands. Recently Linton 21 and others have shown that the secretion of the meibomian glands is unique in that it contains no fatty acids or cholesterol, which are the important prod ucts of all other sebaceous glands. Ac cording to their study, meibomian glands secrete an unidentified lipid that cannot be found in the secretion of sebaceous glands of the skin. This could perhaps be one of the factors determining the abundance of P. orbiculare on the lid margins. 2. MICROSCOPIC EXAMINATION OF SCRAPINGS BUDDING YEASTS IN LID MARGIN SCRAPINGS
Lid margin scrapings are often of great diagnostic value in external diseases of the eye. It was attempted in this study to deter mine whether or not the presence of bud ding yeastlike organisms could be helpful in the diagnosis of blepharitis. Portnoy 22 examined 250 scrapings from the scalp and found P. ovale on 100% of seborrheic and 85% of nonseborrheic scalps. Thygeson and Vaughan 23 found yeastlike organisms on the lid margins of 98%' of pa tients with seborrheic blepharitis and 58% of patients with normal lid margins. In our series of 50 patients with sebor rheic blepharitis, microscopic examination revealed budding yeasts in all scrapings but with a considerable difference in the shape and number of organisms seen. Spherical forms only, or spherical forms mixed with a few oval forms, were seen in 42 scrapings,
APRIL, 1967
oval forms alone in only eight. When scrap ings from 87 patients with normal lid mar gins were examined, spherical forms only or predominantly were seen in scrapings from 81 patients (93%), and no budding yeast like forms were seen in scrapings from the other six patients ( 7 % ) . If, however, we compared the number of organisms seen on seborrheic lid margins with the number seen on normal lid mar gins, yeasts were more numerous on the se borrheic lid margin than on the normal lid margin. But this finding was neither consis tent nor diagnositc. As far as a diagnosis of blepharitis was concerned, the spherical yeast, P. orbiculare, was found in sig nificantly greater numbers only in cases of clinically obvious seborrheic disease. In borderline cases or mild cases, the number of organisms present on the lid margins was of no help in determining etiology. Three of our patients showed very marked scaling, but their lid margins were not inflamed and they had no complaints; P. orbiculare was seen in great numbers in their scrapings and was isolated in culture. Contrary to this, a few patients with clinical evidence of sebor rheic blepharitis, including marked lid mar gin irritation, showed only a few organisms in their lid-margin scrapings, and no isola tions were obtained in culture. SEARCH FOR BUDDING YEASTS AT SPECIAL SITES
1. At the mucocutaneous junction. Vidal claims to have found spherical yeasts in 100% of scrapings from the intramarginal space, that is, between the anterior and pos terior edges of the lid. In an effort to evalu ate his findings, we prepared scrapings from the same area but were either unable to find Pityrosporum or found only a few. 2. In meibomian gland channels. If P. orbiculare lives on the lid margins because of a specific secretion of the meibomian glands, one would expect the yeast to be present in meibomian gland channels. To investigate this possibility, tissues were col-
' VOL. 63, NO. 4
PITYROSPORUM ORBICULARE
lected from the upper tarsal plate of 20 ca davers, three of which had scaly lid mar gins. Histologie sections of these tissues were stained with Gridley stain but no fun gus elements were seen in the gland chan nels. 3. In hair shaft follicles. Spherical yeasts were found surrounding the hair shaft, low in the follicle, when eyelashes were epilated. 3. ANIMAL INOCULATION EXPERIMENTS
Experimental inoculations with either P. ovale or P. orbiculare have failed to produce clinical evidence of seborrhea except under unusual conditions. For example, Moore and his associates5 inoculated a group of seborrheic individuals with P. ovale and re ported 40% positive results. When the ob jection was raised that Moore was produc ing seborrhea in seborrheic individuals, Martin-Scott 6 repeated the experiment with 51 normal, nonseborrheic volunteers and was unable to produce seborrhea. Other at tempts have been made, most of them by an imal inoculation, which have been either un successful or disputable.6·7'9'10'22·24 MATERIALS AND METHODS
In our experiments the inoculum was a pure culture of P. orbiculare isolated from a patient with severe seborrheic blepharitis. Fifteen rabbits were selected as experimental animals. Prior to selection, all animals were examined with the slitlamp. Some of them, interestingly, had fine scales on their lid margins. These were not used for the study but scales from their lid margins were stained with méthylène blue for direct mi croscopic examination and were cultured as already described. No yeastlike organisms were seen or isolated. The rabbits to be inoculated were divided into three groups : Group 1. Six animals were inoculated by rubbing a four-day-old culture on the lower lid of the right eye and on a small shaved area of the back. The sites of inoculation were superficially scarified because previous
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reports stated that a positive result could not be expected unless this was done. The left lid margin was inoculated in the same way except that Saccharomyces cerevisiae, a known nonpathogenic yeast, was used as a control. The culture material was applied both to the skin and to the lid margin once a day for four days. Group 2. Four rabbits received a suspen sion of live P. orbiculare in the conjunctival sac of the right eye, and the control fungus in the conjunctival sac of the left eye, twice a day for two weeks. Group 3. Four rabbits received twice a day for two weeks a cell extract of P. orbic ulare prepared according to the method used to prepare Trichophytin. 25 As a con trol, a fifth animal was given once a day a drop of the medium used for growing P. orbiculare. RESULTS
Material from the lid margins of the rab bits in the first group was collected for mi croscopic examination and culture on the fifth, 12th and 21st days after inoculation. On the fifth day, all scrapings and cultures were positive. On the 12th day, organisms were seen in all scrapings but only two cul tures were positive for P. orbiculare. Final ly, on the 21st day, only a few spherical forms were seen in scrapings from the lid margins of two rabbits; all other scrapings and cultures were negative. Saccharomyces were seen in the scrapings made on the fifth and 12th days. During 30 days of observation, no significant clinical change was observed. There was some scaliness during the first week, but similar changes were also ob served in the controls. The animals in the second and third groups also showed no clinical change dur ing 30 days of observation. SUMMARY AND CONCLUSIONS
1. In an attempt to evaluate the role of the lipophilic yeasts in the etiology of sebor-
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6. Martin-Scott, J. : The Pityrosporum ovale. Brit. J. Dermat. 64:2S7, 1952. 7. Spoor, H. J., Traub, E. F. and Bell, M.: Pi tyrosporum types cultured from normal and se borrheic subjects. Arch. Derm. & Syph. 69:323, 1954. 8. Pillsbury, D. M., Shelley, W. B. and Klingman, A. M. : Textbook of Dermatology. Philadel phia, Saunders, 1956, p. 120. 9. Gotts, J. S., Thygeson, P. and Waisman, M. : Observations on P. ovale in seborrheic blepharitis and conjunctivitis. Am. J. Ophth. 30:1485, 1947. 10. François, J. and Rabley, M. : Etude cytologique et bactériologique des frottis conj. de P. ovale. Ann. Ocul. 183:378, 1950. 11. Vidal, F . : Zeisitis caused by the round Pi tyrosporum. Arch. Oftal. B. Aires, 27 :358, 1952. 12. Duke-Elder, S. : System of Ophthalmol ogy : Vol. Vili. London, Compton, 1965, pp. 397, 546. 13. Feducowitz, H. B. : External infections of the eye. Appleton, New York, 1963, p. 186. 14. Gordon, M. A. : The lipophilic microflora 4. Rabbits were inoculated with both liv of the skin. Mycologia, 43 :524, 1951. ing organisms and cell extracts of P. orbicu 15. Keddie, F. and Shadomy, S. : Etiological lare. Thirty days of observation failed to significance of P. orbiculare in tinea versicolor. Sabouraudia, 3:21, 1963. reveal any significant lesions or changes in 16. Gordon, M. A. : Lipophilic yeast like orga the infected animals. nisms associated with tinea versicolor. Invest. 5. No convincing proof of anything more Derm. 17 :267, 1951. 17. Barfatani, M., Munn, R. and Schjeilde, O. than a casual relationship between Pityro- A. : An ultrastructure study of Pityrosporum or sporum and seborrheic blepharitis could be biculare. J. Invest. Derm. 43 :231, 1964. 18. Spoor, H. J. : A study of antidandruff established. agents. Drug & Cosm. Ind. 77:44, 1955. San Francisco Medical Center (94122) 19. Duke-Elder, S. : System of Ophthalmol ogy: Vol. II. London, Compton, 1961, p. 526. REFERENCES 20. Wolf, E. : Anatomy of the Eye and Orbit. 1. Thygeson, P. : Etiology and treatment of London, Lewis, 1961, ed. 5, p. 173. blepharitis. Arch. Ophth. 36:44S, 1946. 21. Linton, R. G., Curnow, D. H. and Riley, W. 2. Malassez, L. : Note sur le champignon du J. : The Meibomian glands. Brit. J. Ophth. pityriasis simple. Arch. Physiol. 2:4S1, 1874. 45:718, 1961. 3. Benham, R. W. : The cultural characteristics of 22. Portnoy, B. : Seborrheic dermatitis. Med. P. ovale : · A. lipophilic fungus. J. Invest. Derm. 111.4:1,1950. 2:187, 1939. 23. Thygeson, P. and Vaughan, D. G. : Sebor 4. Moore, M. : Cultivation and study of P. rheic blepharitis. Tr. Am. Ophth. Soc. 52:173, ovale, the so-called bottle bacillus of Unna. Arch. 1954. Derm. & Syph. 31:661 1935. 24. Emmons, C. W. : The isolation and pathogen5. Moore, M., Kile, R. M. and Engman, M. F . : icity of P. ovale. Pub. Health Rep. 55:1306, 1940. P. ovale, cultivation and possible role in sebor 25. Muskatblit, E. and Director, W. : The Trichorheic dermatitis. Arch. Derm. & Syph. 33:457, 1936. phytin test. Arch. Derm. & Syph. 27:739, 1933. rheic blepharitis, P. orbiculare Gordon was isolated from the lid margins of 14 seborrheic and five nonseborrheic individuals, ei ther in pure culture or mixed with P. ovale. 2. Parallel cultures and scrapings from the glabrous facial skin and scalp of these same patients showed very few or no P. or biculare. It was concluded that P. orbiculare is more common on the lid margins than on the facial skin or scalp. 3. Microscopic studies of lid margin scrapings showed that P. orbiculare is har bored by as many individuals with normal lid margins as with seborrheic lid margins. It is unlikely, therefore, that P. orbiculare bears a causal relationship to seborrheic blepharitis.